VACCINE FOR IMMUNOCOMPROMISED HOSTS

Abstract

The invention provides peptides derived from a ubiquitous protein, and nucleic acids encoding such peptides. The invention extends to various uses of these peptides and nucleic acids, for example, as antigens for use in vaccines per se and in the generation of antibodies for use in therapeutic drugs for the prevention, amelioration or treatment of infections caused by sepsis-inducing bacteria. The invention particularly benefits immunocompromised hosts such as neonates, babies, children, women of fertile age, pregnant women, foetuses, the elderly and diabetics.

Claims

1. A method of preventing an infection by sepsis-inducing bacteria, the method comprising administering, to a subject in need of such treatment, a peptide that has at least 90% amino acid sequence identity with a peptide found within GAPDH of one or more sepsis-inducing bacteria, or a functional fragment or functional variant thereof.

2. The method of claim 1, wherein the peptide, fragment, or variant has at least 90% amino acid sequence identity with a peptide found within GAPDH of GBS, E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, N. meningitidis and/or Pseudomonas spp.

3. The method of claim 1, wherein the peptide, fragment, or variant has at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity with said peptide found within GAPDH of sepsis-inducing bacteria.

4. The method of claim 1, wherein the peptide, fragment, or variant: a) comprises 150 amino acids or less, or comprises less than 100 amino acids, less than 50 amino acids, less than 30 amino acids or less than 20 amino acids; b) comprises at least 3 amino acids, at least 5 amino acids, at least 8 amino acids or at least 10 amino acids; and/or c) is 5-100 amino acids in length, 5-50 amino acids in length or 10-20 amino acids in length.

5. The method of claim 1, wherein the peptide, fragment, or variant has at least 90% amino acid sequence identity with an amino acid sequence found within any one of SEQ ID NOs: 1 to 7, and is at least 8 amino acids and less than 50 amino acids in length.

6. The method of claim 1, wherein the peptide, fragment, or variant has at least 90% amino acid sequence identity with any one of SEQ ID NOs: 9-69, and is at least 8 amino acids and less than 50 amino acids in length.

7. The method of claim 1, wherein the peptide, fragment, or variant has at least 90% amino acid sequence identity with an amino acid sequence found within SEQ ID NO: 3, is at least 8 amino acids and less than 50 amino acids in length, and comprises an amino acid sequence with at least 95% amino acid sequence identity to any one of SEQ ID NOs: 26-33.

8. The method of claim 1, wherein the peptide, fragment, or variant has at least 90% amino acid sequence identity with an amino acid sequence found within SEQ ID NO: 4, is at least 8 amino acids and less than 50 amino acids in length, and comprises an amino acid sequence with at least 95% amino acid sequence identity to any one of SEQ ID NOs: 34-41.

9. The method of claim 1, wherein the peptide, fragment, or variant has an amino acid sequence substantially as set out in any one of SEQ ID NOs: 9-69.

10. The method claim 1, wherein the peptide, fragment, or variant is conjugated to a carrier protein.

11. The method claim 6, wherein the peptide, fragment, or variant is conjugated to a carrier protein.

12. The method claim 7, wherein the peptide, fragment, or variant is conjugated to a carrier protein.

13. The method claim 8, wherein the peptide, fragment, or variant is conjugated to a carrier protein.

14. The method claim 9, wherein the peptide, fragment, or variant is conjugated to a carrier protein.

15. The method of claim 1, wherein the method is a method of preventing any one or more of sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, encephalitis, premature birth and stillbirth.

16. The method of claim 1, wherein the subject in need of treatment is an immunocompromised host.

17. The method of claim 16, wherein the immunocompromised host is selected from a neonate, baby, child, woman of fertile age, pregnant woman, foetus, diabetic and/or an elderly subject.

18. The method of claim 1, wherein the sepsis-inducing bacteria are selected from the group consisting of GBS, E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, Pseudomonas spp. and N. meningitidis.

Description

[0184] 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, by way of example, to the accompanying diagrammatic drawings, in which:

[0185] FIG. 1 shows an amino acid sequence alignment for GAPDH of the main sepsis-inducing bacteria, GBS, E. coli, S. aureus, S. pneumoniae, K. pneumoniae, P. aeruginosa and N. meningitidis. The multiple alignment was obtained from the ClustalW2 server, after submitting the amino acid sequences identified herein as SEQ ID NOs: 1-7 (according to the FASTA format of the previously indicated UniProt accession numbers). The resultant % sequence similarities are shown in Table 1.

[0186] FIG. 2 provides an example of four surface peptides that can be used in a vaccine of the invention. The table shows the amino acid sequences of the four exemplary peptides, and the respective bacteria that possess each amino acid sequence. Below the table is indicated the surface localisation of the same four peptides in the different bacterial GAPDHs. The peptides are identified herein as Peptides 1-4 (SEQ ID NOs: 9-12), and they are used in combination to form the vaccine identified herein as Neonatal Vaccine.

[0187] FIG. 3 shows an alignment of the amino acid sequence of GAPDH from S. agalactiae (SEQ ID NO:70), S. pneumoniae (SEQ ID NO:4), S. aureus (SEQ ID NO:3) and humans (SEQ ID NO:8). The two boxed areas show the sequences from which Peptide 1 (SEQ ID NO:9) and Peptide 2 (SEQ ID NO:71) are derived.

[0188] FIGS. 4-6 show that neonatal B1 cells are the major producers of IL-10 upon stimulus by bacterial GAPDH. Panels A and B of FIG. 4 show IL-10 concentration following the stimulation of spleen mononuclear cells (MNC), neutrophils from peripheral blood (PMNC), macrophages (FLM) or dendritic cells (FLDC) derived from the liver and B cells (total), B1 cells and B2 cells purified from the spleen of newborn mice with lipopolysaccharide (LPS), rGAPDH or Roswell Park Memorial Institute (RPMI) medium alone. FIG. 5 shows IL-10 concentration following the stimulation of B1 cells purified from the spleen of newborn mice with rGAPDH in the presence of a TLR2 inhibitor (OxPAC) or Toll-like receptor 4 (TLR4) inhibitor (CLI095) as shown. Panels A and B of FIG. 6 show IL-10 concentration following the stimulation of total cells and B1 cells, respectively, purified from the spleen of newborn mice with rGAPDH, fixed GBS (GBSf) or RPMI medium alone. Panel C of FIG. 6 shows IL-10 concentration following the stimulation of a co-culture of dendritic cells derived from foetal liver and B1 cells purified from newborn spleen with rGAPDH, GBSf, a monoclonal antibody specific for type I interferon receptor (aIFNAR) or RPMI medium alone. Data depicted in all of the panels for FIGS. 4-6 are mean+SEM of at least two independent experiments.

[0189] FIG. 7 shows that TLR2 deficiency improves neonatal survival and confers protection to bacterial sepsis. The survival of newborn mice challenged with S. aureus strain NEWMAN (panel A) or E. coli strain IHE3034 (panel B) is shown. Both wild-type and TLR2.sup.?/? mice were included in the study. Results represent data pooled from at least two independent experiments. The numbers between parentheses represent the number of animals that survived the different infectious challenges versus the total number of infected animals. Statistical differences (P values) between TLR2-deficient pups versus controls are indicated.

[0190] FIG. 8 shows that blocking IL-10 signaling protects newborns from bacterial sepsis. The survival of newborn mice challenged with E. coli strain IHE3034 (panel A) or S. aureus strain NEWMAN (panel B) following an injection of monoclonal antibodies specific for the mouse IL-10 receptor (anti-IL10R) or isotype-matched control antibodies (isotype IgG/control) is shown. Results represent data pooled from three independent experiments. The numbers between parentheses represent the number of animals that survived the different infectious challenges versus the total number of infected animals. Statistical differences (P values) between anti-IL-10R-treated pups versus controls are indicated.

[0191] FIG. 9 shows that GAPDH secretion is a shared virulence mechanism. rGAPDH, which was used as a positive control, is shown in lane 1. Lanes 2-6 show an equivalent band for the indicated pathogens (NEM316 being a strain of GBS). The data are representative of five independent experiments.

[0192] FIG. 10 shows that antibodies elicited with rGAPDH protect newborn mice from infection by sepsis-inducing bacteria other than GBS. The survival of mice pups challenged with S. pneumonia strain Tigr4 (Panel A), E. coli strain IHE3034 (Panel B) and S. aureus strain NEWMAN (Panel C) following an injection of rGAPDH-induced antibodies (anti-rGAPDH IgG) or control antibodies (Control IgG) is shown. Results represent data pooled from two independent experiments. The numbers between parentheses represent the number of animals that survived the various infectious challenges versus the total number of infected animals. Statistical differences (P values) between immunised versus control groups are indicated.

[0193] FIG. 11 shows that bacterial GAPDH induces IL-10 production in human mononuclear cells. Panels A and B show IL-10 concentration following the stimulation of human mononuclear cells separated from cord-blood (panel A) or peripheral blood (panel B) with rGAPDH, a TLR2 inhibitor (TLR2 in) or RPMI medium alone. Data depicted in the figure are mean+SEM of at least two independent experiments.

[0194] FIG. 12 shows an alignment of the amino acid sequence of GAPDH from E. coli (SEQ ID NO:72) and humans (SEQ ID NO:8). The boxed area shows Peptide 3 from FIG. 2. On the bottom right of FIG. 12, Peptide: EVKDGHLIVNGKK (SEQ ID NO:73).

[0195] FIG. 13 shows an alignment of the amino acid sequence of GAPDH from P. aeroginosa (SEQ ID NO:6) and humans (SEQ ID NO:8). The boxed area at amino acid 23 shows a peptide for use in the invention (SEQ ID NO: 62). The boxed area at amino acid 59 shows Peptide 4 from FIG. 2. On the bottom of FIG. 12, Peptide 1: TGHYREQLQ (SEQ ID NO:62); and Peptide 2: EHDAESLRVMGDR (SEQ ID NO:12).

[0196] FIGS. 14, 15 and 16 show that antibodies elicited with a vaccine of the invention react with bacterial GAPDH. rGAPDH, which was used as a positive control, is shown in lane 1 of each gel. Lanes 2-6 in FIG. 14 and lane 2 in FIGS. 15 and 16 show an equivalent band for the indicated pathogens. The data are representative of two independent experiments.

[0197] FIG. 17 shows that antibodies elicited with Neonatal Vaccine protect newborn mice from GBS infection. The survival of mice pups challenged with GBS NEM316 following an injection of Neonatal Vaccine-induced antibodies (IgG) or control IgG is shown.

[0198] Results represent data pooled from two independent experiments. The numbers between parentheses represent the number of animals that survived the infectious so challenge versus the total number of infected animals. Statistical differences (P values) between immunised versus control groups are indicated.

[0199] FIG. 18 shows that antibodies elicited with Neonatal Vaccine protect newborn mice from bacterial sepsis. The survival of newborn mice challenged with S. pneumoniae strain Tigr4 (panel A), E. coli strain IHE3034 (panel B) or S. aureus strain NEWMAN (panel C) following an injection of Neonatal Vaccine-induced antibodies (IgG) or control IgG is shown. Results represent data pooled from at least two independent experiments. The numbers between parentheses represent the number of animals that survived the different infectious challenges versus the total number of infected animals. Statistical differences (P values) between immunised versus controls are indicated.

[0200] FIG. 19 shows that the therapeutic use of anti-GAPDH antibodies can efficiently treat GBS-induced sepsis. The survival of newborn mice challenged with GBS NEM316 and subsequently receiving Neonatal Vaccine-induced antibodies (IgG), control IgG or saline is shown. Results represent data pooled from two independent experiments. The numbers between parentheses represent the number of animals that survived the infectious challenge versus the total number of infected animals. Statistical differences (P values) between immunised versus controls are indicated.

[0201] FIG. 20 shows that antibodies elicited with Neonatal Vaccine protect old mice against lethal GBS infection. The survival of old mice challenged with GBS following an injection of Neonatal Vaccine-induced antibodies (Neonatal Vaccine-IgG) or control IgG (sham-immunized) is shown. Results represent data pooled from two independent experiments. The numbers between parentheses represent the number of animals that survived the infectious challenge versus the total number of infected animals. The statistical difference (P value) between immunised versus control groups is indicated.

[0202] FIG. 21 shows that antibodies elicited with Neonatal Vaccine protect non-obese diabetic (NOD) mice against lethal GBS infection. The survival of NOD mice challenged with GBS following injections of Neonatal Vaccine-induced antibodies (Neonatal Vaccine-IgG) or control IgG (sham-immunized) is shown. Results represent data pooled from two independent experiments. The numbers between parentheses represent the number of animals that survived the infectious challenge so versus the total number of infected animals. The statistical difference (P value) between immunised versus control groups is indicated.

EXAMPLES

[0203] The materials and methods employed in the studies described in the Examples were as follows, unless where otherwise indicated:

Mice

[0204] Six- to eight-week-old male and female BALB/c, C57BL/6, and TLR2- deficient C57BL/B6.129-Tlr2.sup.tm1Kir/J (TLR2.sup.?/?) mice, and old C57Bl/6 mice (over 16 months), were purchased from The Jackson Laboratory. New Zealand White rabbits and eight-week-old non-obese diabetic (NOD) mice were purchased from Charles River Laboratories. Animals were kept at the animal facilities of the Institute Abel Salazar during the time of the experiments. All procedures were performed according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS 123) and 86/609/EEC Directive and Portuguese rules (DL 129/92). All animal experiments were planned to minimise animal suffering.

Bacteria

[0205] The bacteria used in the studies are listed in Table 4 below. All strains were clinical isolates obtained from infected newborns. E. coli, S. aureus, P. aeruginosa, GBS and S. pneumoniae were kindly provided by Professor Patrick Trieu Cuot from Pasteur Institute, Paris, France; K. pneumoniae and N. meningitidis were provided by the Microbiology Department of Hospital Geral de Santo Ant?nio, Porto, Portugal. GBS and S. pneumoniae were grown in Todd-Hewitt broth or agar (Difco Laboratories) containing 0.001 mg/mL of colistin sulphate and 0.5 ?g/mL of oxalinic acid (Streptococcus Selective Supplement, Oxoid). E. coli, P. aeruginosa, MenB and S. aureus were cultured on Todd-Hewitt broth or agar medium. Bacteria were grown at 37? C.

TABLE-US-00016 TABLE 4 Bacteria used in the studies described in the Examples Bacteria Strain Escherichia coli IHE.sub.3034 Staphylococcus aureus NEWMAN Pseudomonas aeroginosa PAO.sub.4 Streptococcus agalactiae, GBS NEM.sub.316 Streptococcus pneumoniae Tigr.sub.4 Neisseria meningitidis Serogroup B (MenB)

Antibody Treatments

[0206] Antibody treatments were performed in newborn BALB/c mice (up to 48 h old) 12 h prior to GBS infection, and in old C57Bl/6 mice (over 16 months) and NOD mice 24 h prior to GBS infection. For passive immunisations, pups were intraperitoneally injected with 100 ?g of anti-rGAPDH IgG antibodies. Control animals received the same amount of control IgG antibodies. For IL-10 signaling blocking, 100 ?g of anti-IL10R antibodies (1B1.3a, Schering-Plough Corporation) were administered intraperitoneally and control animals received the same amount of matched isotype control antibody. Regarding the therapeutic use of anti-GAPDH antibodies, mice pups were treated with 100 ?g of anti-GAPDH IgG (or the respective control IgG) 6h after infection.

Neonatal Mouse Model of Bacterial Infection

[0207] Neonatal (48 h old), BALB/c, C57BL/6 wild-type or TLR2.sup.?/? mice were infected subcutaneously with the indicated inoculum of the bacteria in a maximum volume of 40 ?l. Newborns were kept with their mothers during the entire time of the experiment. Survival curves were determined over a 12-day experimental period.

rGAPDH

[0208] rGAPDH was produced and purified as previously described [41].

Purification of Anti-GAPDH IgG

[0209] Adult mice or rabbits were immunised twice with 25 ?g of rGAPDH in a PBS/alum suspension with a three-week interval between doses. Sera were collected 10 days after the second immunisation. Pooled serum samples were applied to a Protein G HP affinity column (HiTrap, GE Healthcare Bio-Sciences AB) and purified IgG antibodies were then passed through an affinity column with immobilised rGAPDH (Hi-trap NHS-activated HP, GE Health-care Bio-Sciences AB). Control IgGs were obtained from sera of mice or rabbits sham-immunised with a PBS/alum suspension and purified on a Protein G HP affinity column. Purified IgG antibody fractions were further equilibrated in PBS and stored at ?80? C. in frozen aliquots.

Spleen Total Cell Cultures

[0210] Cells from the spleen of newborn mice (up to 48 h old) were obtained by gently teasing the organ in RPMI 1640 supplemented with penicillin (100 IU/ml), streptomycin (50 ?g/ml), 2-ME (0.05 M), and 10% foetal bovine serum (FBS) (Sigma-Aldrich)-complete RPMI (cRPMI). Cells were then distributed in 96-well plates (1?10.sup.6 cells/well) and cultured for 12 h at 37? C. in a humidified atmosphere containing 5% carbon dioxide, with the medium alone, medium containing 2.5 ?g/ml LPS, medium containing 25 ?g/ml of rGAPDH, medium containing 1 ?g/mL of the TLR2 agonist, PAM3CSK4 (Invivogen). For the experiments with the TLR inhibitors, OxPAC (TLR2 inhibitor) and CLI095 (TLR4 inhibitor) (both from Invivogen) were used at a concentration of 10 ?g/mL.

B Cell Purification

[0211] B cells were purified from the spleen of neonatal mice (prepared as mentioned above) by magnetic cell sorting using a Mouse B cell Purification Kit (Miltenyi Biotech) according to manufacturer's instructions.

CD5.sup.+ B cell purification

[0212] B1 cells were purified from the spleen of neonatal mice (prepared as mentioned above) by magnetic cell sorting, using a Mouse B1 cell Purification Kit (Miltenyi Biotech) according to manufacturer's instructions.

Neonatal Liver-Derived Macrophages

[0213] Macrophages were obtained from the liver of one-day old mice. Livers were removed under aseptic conditions and homogenised in Hanks' balanced salt solution (HBSS). The resulting cell suspension was centrifuged at 500?g and resuspended in cRPMI supplemented with 10% L929 cell conditioned medium. To remove fibroblasts or differentiated macrophages, cells were cultured, on cell culture dishes, overnight at 37? C. in a 5% carbon dioxide atmosphere. Then, non-adherent cells were collected with warm cRPMI, centrifuged at 500?g, distributed in 96-well plates at a density of 1?10.sup.5 cells/well, and incubated at 37? C. in a 5% carbon dioxide atmosphere. Four days after seeding, 10% of L929 cell conditioned medium was added, and the medium was renewed on the seventh day. After 10 days in culture, cells were completely differentiated into macrophages. This method allows for the differentiation of a homogenous primary culture of macrophages that retain the morphological, physiological and surface markers characteristics of these phagocytic cells [50].

Neonatal Liver-Derived Dendritic Cells

[0214] Dendritic cells were obtained from the liver of one-day old mice. Livers were removed under aseptic conditions and homogenised in HBSS. The resulting cell suspension was centrifuged at 500?g and resuspended in cRPMI supplemented with 30 ng/ml of granulocyte macrophage colony-stimulating factor (GM-CSF) (Immunotools) (Primary DC media). To remove fibroblasts or differentiated macrophages, cells were cultured, on cell culture dishes, overnight at 37? C. in a 5% carbon dioxide atmosphere. At day 3, 75% of the medium (along with non-adherent cells) was removed, and Primary DC media was added. At day 6, cells were removed from the plate by gently pipetting media up and down against the bottom of the plate to gently dislodge non-adherent cells. After several minutes of this, the cell mixture was transferred to 50 mL polystyrene tubes. Cells were then centrifuged at 500?g for 5-7 min and re-suspended in Primary DC media. The cells were counted and plated at a concentration of 5?10.sup.5 cells/well. For the co-culture experiments, 5?10.sup.4 dendritic cells were plated per well. In the co-culture experiments, and where indicated, 20 ?g/mL of a monoclonal antibody specific for Type I interferon receptor (anti-IFNAR) (Biolegend) was used.

Purification of Blood Neutrophils

[0215] For neutrophil isolation, blood was collected from retro-orbital bleeding of neonatal mice (up to 48 h old) and diluted 1:2 in HBSS containing BSA (0.1% w/v) and glucose (1% w/v). Cells were pelleted, and erythrocytes were removed by hypotonic lysis. The blood preparation was suspended in Dulbecco's PBS (GIBCO), layered on a three-layer Percoll (GE-Healthcare) gradient (80, 65, and 55% in Dulbecco's PBS), and centrifuged at 1200?g for 30 min at 10? C. Mature neutrophils were recovered at the interface of the 65 and 80% fractions, and purity was 85%, as determined by FACS analysis, using anti-Ly6G antibodies (Biolegend). Isolated neutrophils were plated on 96-well plates and stimulated for 12 h as indicated.

IL-10 Quantification

[0216] IL-10 from newborn or adult cell cultures was quantified by ELISA (R&D Systems), according to the manufacturer's instructions.

Human Blood Samples

[0217] Human blood samples were obtained at Hospital Geral de Santo Ant?nio after informed approval. For the isolation of mononuclear cells, 5 ml aliquots of total blood diluted 1:2 in RPMI 1640 were layered on 2.5 ml of Histopaque (Sigma-Aldrich) and centrifuged at 1000 g for 20 min at room temperature. The cells were then gently removed from the medium-Histopaque interface, transferred to a sterile container, and washed in 10 ml of cRPMI. The isolated mononuclear cells were re-suspended in cRPMI, plated at a concentration of 5?10.sup.5 cells/well and stimulated with 25 ?g/mL of rGAPDH, with 10 ?g/mL of OxPAC or with medium alone (RPMI) for 12 h at 37? C. with 5% carbon dioxide.

Neonatal Vaccine

[0218] Peptides 1-4 (SEQ ID NOs: 9-12) were conjugated with KLH or OVA as carrier proteins. For the immunisation protocols, 20 ?g of each peptide conjugated with the carrier protein was injected intraperitoneally in female BALB/c mice. Alum was used as adjuvant in a 1:20 PBS suspension. Adult female BALB/c mice were immunised three times with a three-week interval between doses. 10 days after the last immunisation, blood was collected and the Neonatal Vaccine anti-serum was obtained after blood clotting at 4? C. for 24 hours.

[0219] The same immunisation protocol was used in rats for the N. meningitidis work (Example 8).

Example 1a Sub-Population of Neonatal B Cells is Responsible for IL-10 Production Upon Bacterial GAPDH Stimulus

[0220] Previous published information revealed the role of GAPDH in disabling the neonatal immune system to combat GBS infections [24]. Although a role for IL-10 was already unveiled in the mechanism of GAPDH-induced immunosuppression, the cellular mechanism remained unknown. In order to uncover which cellular population(s) was contributing to early IL-10 production observed in neonatal GBS infections, different leukocyte populations were purified from neonatal mice and treated in vitro with rGAPDH from GBS.

Materials and Methods

[0221] Specifically, and as described above, dendritic cells and macrophages were obtained from neonatal liver precursors, B cells and mononuclear cells were obtained from neonatal spleens and neutrophils were purified from neonatal peripheral blood. A more refined separation of B cells obtained from neonatal spleen, based on surface expression of CD5, allowed the separation of B1 (CD5+) cells.

[0222] The different leukocyte populations were stimulated in vitro with 0.5 ?g/mL of LPS (as a positive control; LPS is a structural microbial antigen known to induce polyclonal B cell activation), 25 ?g/mL of rGAPDH or RPMI medium alone (as a negative control) for 12 h at 37? C. with 5% carbon dioxide. In all conditions 5?10.sup.5 cells/well were used, except for the separated B cell study, where 2.5?10.sup.5 cells/well were used.

[0223] After incubation of the cells, IL-10 concentration was measured in the supernatants as described above.

[0224] At least two independent experiments were performed in each case.

Results

[0225] As observed in FIG. 4A, when comparing the ability of mononuclear cells, neutrophils, macrophages, dendritic cells and total B cells to produce IL-10 upon GAPDH stimulus, only B cells retained the ability to produce significant amounts of IL-10.

[0226] Following the separation of neonatal B cells, the inventors observed that B1 cells retained the ability to produce IL-10 while B2 cells produced only traceable amounts of this cytokine (FIG. 4B).

Discussion

[0227] This study indicates that neonatal B1 cells are the main source of IL-10 upon bacterial GAPDH stimulus.

Example 2TLR2 is the Surface Receptor for Bacterial GAPDH

[0228] In order to establish the cellular receptor responsible for bacterial GAPDH recognition and induction of IL-10 expression, the inventors compared the ability of GAPDH to induce IL-10 production in cultures of purified B1 cells in the presence of specific inhibitors of different pattern recognition receptors.

Materials and Methods

[0229] B1 cells were purified from the spleen of newborn mice as described above. 2.5?10.sup.5 B1 cells/well were stimulated in vitro with 25 ?g/mL of rGAPDH in the presence of 10 ?g/ml of TLR2 or TLR4 inhibitors for 12 h at 37? C. with 5% carbon dioxide. The TLR2 and TLR4 inhibitors used were OxPAC and CLI095, respectively.

[0230] After incubation of the cells, IL-10 was quantified in the supernatants as described above.

[0231] At least two independent experiments were performed.

Results

[0232] The inventors found that GAPDH-induced IL-10 production was completely abrogated in the presence of a TLR2 inhibitor (FIG. 5).

Discussion

[0233] This result indicates that bacterial GAPDH acts on B1 cells through TLR2 in order to induce IL-10 production.

Example 3TLR2 Deficiency Improves Neonatal Survival and Confers Protection to Bacterial Sepsis

[0234] This study aimed to confirm the importance of TLR2 as a receptor for GAPDH, and a cause for neonatal susceptibility to sepsis.

Materials and Methods

[0235] 48 hours after birth, newborn wild-type and TLR2.sup.?/? mice were infected subcutaneously with 5?10.sup.5 CFU of S. aureus strain NEWMAN or with 500 CFU of E. coli strain IHE3034. Survival of the mice following infection was monitored on a daily basis.

[0236] At least two independent experiments were performed.

Results

[0237] Wild-type mice were unable to survive infection with the indicated bacteria beyond 48 hours post-infection (FIG. 7). In contrast, the majority of TLR2?/? mice were still alive at 12 days after infection.

Discussion

[0238] The results shows that TLR2-deficient neonatal mice have increased survival against challenging infections with E. coli and S. aureus compared to wild-type mice. TLR2 thus plays an important role in neonatal susceptibility to sepsis; that is to say, TLR2 deficiency improves neonatal survival and confers protection to bacterial sepsis. In addition to the results obtained in Example 2, these data thus confirm the importance of TLR2 as a receptor for GAPDH, across the different species of sepsis-inducing bacteria.

Example 4Type I Interferon Production by Dendritic Cells Induced by Bacteria Synergises with GAPDH to Increase IL-10 Production on B1 Cells

[0239] This study aimed to identify whether B1 cells are assisted in the production of IL-10 upon GAPDH recognition by other leukocyte populations, and in what capacity.

Materials and Methods

[0240] Total spleen cells were obtained from newborn mice, and B1 cells were purified from the total spleen cell population, as described above.

[0241] The different spleen cell populations were stimulated in vitro with 25 ?g/mL of rGAPDH, 10.sup.7 cells of GBS fixed in isopropanol (GBSf) or with RPMI medium alone for 12 h at 37? C. with 5% carbon dioxide. In all conditions 5?10.sup.5 cells/well were used, except for the purified B1 cell study where 2.5?10.sup.5 cells/well were used.

[0242] Dendritic cells were derived from foetal liver as described above. The dendritic cells were co-cultured with 2.5?10.sup.5 of the B1 cells purified from newborn spleen in a 1:10 ratio and stimulated with 25 ?g/mL of rGAPDH, 10.sup.7 cells of GBSf, 20 ?g/mL of anti-IFNAR or with RPMI medium alone for 12 h at 37? C. with 5% carbon dioxide.

[0243] After incubation of the different cell types, IL-10 was quantified in the supernatants as so described above.

[0244] At least two independent experiments were performed in each case.

Results & Discussion

[0245] The ability of GAPDH to induce IL-10 production in total spleen cells was strongly increased in the presence of fixed bacteria (FIG. 6A). Nevertheless, this effect was lost in purified B1 cells, where adding fixed bacteria did not increase the IL-10 production induced by GAPDH (FIG. 6B).

[0246] This result indicates that different leukocyte population(s) other than B1 cells are stimulated by bacterial antigens and help B1 cells to produce IL-10 upon GAPDH recognition.

[0247] The co-culture study enabled understanding of the role of other sub-populations of leukocytes in the influence of B1 cells to produce IL-10. The inventors observed that in the presence of dendritic cells, B1 cells produced elevated amounts of IL-10 when stimulated simultaneously with GAPDH plus GBSf (FIG. 6C). Interestingly, this effect was abrogated when type I interferon signalling was blocked (FIG. 6C).

[0248] This result indicates that upon bacterial recognition, dendritic cells produce type I interferon that increase IL-10 production in B1 cells stimulated with GAPDH.

Example 5Early IL-10 production is a generalised mechanism used by sepsis-inducing bacteria to colonise the neonatal host

[0249] The ability to produce high amounts of IL-10 was demonstrated to be the main reason for the susceptibility of neonates against GBS infections [24]. The present study aimed to investigate whether the same happens in neonatal infections caused by bacteria other than GBS, specifically E. coli or S. aureus. Together with GBS, E. coli and Staphylococcal spp. are responsible for up to 87% of the cases of sepsis in human neonates.

[0250] The study also aimed to investigate whether other bacteria also possess extracellular GAPDH, as an indication of a generalised IL-10-dependent mechanism used by sepsis-inducing bacteria to colonise the neonatal host.

Materials and Methods

[0251] Neonatal mice were treated with blocking antibodies specific for the mouse IL-10 receptor (anti-IL10R) before challenge with E. coli or S. aureus, as follows.

[0252] Newborn mice were intraperitoneally injected with 100 ?g of anti-IL10R monoclonal antibodies or 100 ?g of isotype control IgG as described above. 12 h later the mice were challenged subcutaneously with 500 CFU of E. coli strain IHE3034 or with 5?10.sup.5 CFU of S. aureus strain. Survival of the mice following infection was monitored on a daily basis.

[0253] Three independent experiments were performed.

[0254] To investigate whether other sepsis-inducing pathogens also possess extracellular GAPDH, extracellular proteins from culture supernatants of pathogens of interest were obtained, separated by SDS-PAGE and analysed by western-blot using anti-rGAPDH antibodies, as follows.

[0255] Cultures of GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus and E. coli were prepared as described above, and extracellular proteins purified from the culture supernatants in accordance with standard procedures. SDS-PAGE and Western-blot analysis were performed according to standard procedures using anti-rGAPDH antibodies (IgG) obtained from rGAPDH-immunised rabbits as described above. rGAPDH was used as a positive control.

[0256] Five independent experiments were performed.

[0257] The effect of neutralising GAPDH secreted by these bacteria, to assess whether these pathogens also use GAPDH secretion as a virulence mechanism, was investigated as follows.

[0258] Mice pups were intraperitoneally injected with 80 ?g of anti-rGAPDH antibodies (IgG) or 80 ?g of control IgG as described above. 12 h later the mice were challenged subcutaneously with 5?10.sup.6 CFU of S. pneumoniae strain Tigr4, 500 CFU of E. coli strain IHE3034 or 5?10.sup.5 CFU of S. aureus strain NEWMAN. Survival of the mice following infection was monitored on a daily basis.

[0259] Two independent experiments were performed.

Results

[0260] Interestingly, blocking IL-10 signalling significantly improved survival of neonates to infections caused by E. coli and S. aureus, when compared with pups that received isotype-matched control antibodies (FIG. 8).

[0261] Other sepsis-inducing bacteria were also shown to possess extracellular GAPDH (FIG. 9). Neutralisation of this secreted GAPDH using anti-rGAPDH antibodies was shown to protect newborn mice from infection by S. pneumoniae, E. coli and S. aureus (FIG. 10A-C, respectively).

Discussion

[0262] The results indicate that the mechanism observed for the susceptibility of neonates against GBS-induced sepsis is transversal to other sepsis-inducing bacteria. Although IL-10 data for neonatal infections caused by E. coli and S. aureus are provided here, the fact that other bacteria also secrete GAPDH is a strong indicator that the propensity of neonates to produce high amounts of IL-10 in response to bacterial GAPDH is a global mechanism used by different bacterial pathogens, which leads to the development of sepsis. This result validates the fact that a GAPDH-based vaccine against GBS will also be viable against other sepsis-inducing bacteria too.

Example 6GAPDH-Induced IL-10 Production is a Mechanism Conserved in Human Cells

[0263] This study aimed to investigate whether human cells also produce IL-10 in response to GAPDH.

Materials and Methods

[0264] Mononuclear cells were separated from human cord-blood or adult peripheral blood as described above.

[0265] The cells were stimulated in vitro with 25 ?g/mL of rGAPDH, 10 ?g/mL of OxPAC (a TLR2 inhibitor) or RPMI medium alone for 12 h at 37? C. with 5% carbon dioxide.

[0266] After incubation of the cells, IL-10 was quantified in the supernatants as described above.

[0267] At least two independent experiments were performed.

Results

[0268] In agreement with what was observed in neonatal mice, the stimulation of mononuclear cells purified from human cord-blood or adult peripheral blood with rGAPDH induced the production of high amounts of IL-10 (FIG. 11A and FIG. 11B, respectively).

[0269] Interestingly, GAPDH-induced IL-10 production in human leukocytes was completely abrogated in the presence of a TLR2 inhibitor.

Discussion

[0270] This result shows that the mechanism for IL-10 production induced by GAPDH in mouse cells is also true for humans.

[0271] Moreover, the fact that the same virulence mechanism studied in mice can be readily translated to humans strongly supports the use of mice as an excellent model to study bacterial sepsis in man.

Example 7Production of Neonatal Vaccine

[0272] Based on their discovery that the propensity of neonates to produce high amounts of IL-10 in response to bacterial GAPDH is a global mechanism used by different bacterial pathogens, the inventors set out to produce a vaccine against such pathogens using GAPDH-derived peptides as the antigen.

Materials and Methods

[0273] Neonatal Vaccine was prepared as described above.

Results

[0274] Vaccines of the invention are composed from surface peptides of GAPDH from the different sepsis-inducing bacteria, which have amino acid sequences that are absent from human GAPDH. As such, the inventors have developed vaccines composed of peptides belonging to conserved sequences of microbial GAPDH that are not shared by human GAPDH.

[0275] FIG. 2 includes a table identifying the amino acid sequences of four exemplary peptides that were found in the present study, using the above method, and the respective bacteria that possess each amino acid sequence. Below the table are images showing the surface localisation of the same four peptides in the different bacterial GAPDHs.

[0276] FIG. 3 illustrates how two of the peptides (identified as Peptides 1 and 2 in FIGS. 2 and 3) have amino acid sequences which are conserved amongst certain bacterial species, but not in humans.

[0277] FIGS. 12 and 13 illustrate how two further peptides from E. coli and P. aeruginosa (identified as Peptides 3 and 4, respectively, in FIG. 2) have (bacterially conserved) amino acid sequences which are not found in man.

[0278] Peptides 1-4 were used in combination in the preparation of a preferred vaccine of the invention, referred to herein as Neonatal Vaccine. Thus, Neonatal Vaccine is suitable for use against all of the bacteria listed in the table in FIG. 2.

[0279] Peptides 1-4, however, are mere examples; that is to say, other peptides that are suitable for use in a vaccine of the invention can be identified by sequence alignment in the same way as set out above. Any other amino acid sequence can be used from GAPDHs of the referred pathogens. As explained herein, however, it is preferable to avoid any sequences that are also found in man, so as to avoid any autoimmune pathologies.

[0280] As described herein, any number of peptides, in any combination, can be used instead of Peptides 1-4 of Neonatal Vaccine. That is to say, the number and identity of peptides that constitute a vaccine of the invention can vary.

Discussion

[0281] As explained herein, bacterial GAPDH plays a role in causing immunosuppression in neonates and immunocompromised hosts and promoting bacterial sepsis. The vaccines described herein, including the specific Neonatal Vaccine described in this example, are thus directed to protect susceptible hosts (including neonates, the elderly and other such immunocompromised individuals) from infections caused by GBS, E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae and Pseudomonas spp. The approach taken by the inventors allows the possibility to tailor a vaccine of the invention for any sepsis-inducing bacteria, simply by selecting the surface-exposed peptides of its GAPDH that are absent from human GAPDH.

Example 8Antibodies Elicited with Neonatal Vaccine React with Bacterial GAPDH

[0282] This study aimed to show that the vaccine produced in Example 7 could be used to produce antibodies that recognise bacterial GAPDH.

Materials and Methods

[0283] Mice and rats were immunised with Neonatal Vaccine as described above.

[0284] Cultures of GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and MenB were prepared as described above, and extracellular proteins purified from the culture supernatants in accordance with standard procedures. SDS-PAGE and Western-blot analysis were performed according to standard procedures using anti-GAPDH antibodies (IgG) obtained from the rGAPDH-immunised mice and rats as described above. rGAPDH was used as a positive control.

[0285] Two independent experiments were performed.

Results

[0286] As shown in FIGS. 14-16, antibodies purified from mice and rats immunised with Neonatal Vaccine react with extracellular GAPDH from the different bacteria. FIG. 15 (showing results from K. pneumoniae) reveals two bands that are recognised by anti-GAPDH antibodies elicited with Neonatal Vaccine. Interestingly, the band of ?45 KDa corresponds to exactly the same molecular weight of GBS GAPDH. The other band (?35 KDa) corresponds to the predicted molecular weight of K. pneumoniae GAPDH (http://www.uniprot.org/uniprot/C4X7S6).

[0287] FIG. 16 (showing results from MenB) reveals a band of ?37 kDa, which corresponds to the predicted molecular weight of MenB GAPDH [51].

Discussion

[0288] This study shows that antibodies elicited with Neonatal Vaccine recognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and MenB. These data therefore provide proof-of-concept that bacterial peptide sequences in common can be used in a vaccine to recognise bacterial GAPDH.

[0289] Although only serotype B of N. meningitidis has been tested here, similar results would be expected for all other serotypes of this bacterium. In this regard, GAPDHs from the different serotypes of N. meningitidis share high (97.668%) homology (http://www.uniprot.org/align/A20150610146R80D4XR) and antibodies elicited with Neonatal Vaccine would therefore be expected to recognise GAPDH from them all. It is consequently believed that the vaccines described herein are advantageous for all the serotypes of N. meningitidis.

[0290] The result illustrated in FIG. 15 also shows that, interestingly, K. pneumoniae may possess two isoforms of GAPDH.

Example 9Neonatal Vaccine Protects Neonates from GBS Infection

[0291] This study aimed to show that the antibodies produced in Example 8 could be used to protect newborn mice from GBS infection.

Materials and Methods

[0292] Mice pups were intraperitoneally injected with 80 ?g of Neonatal Vaccine-induced IgG or 80 ?g of control IgG as described above. 12 h later the mice were challenged subcutaneously with 5?10.sup.6 CFU of GBS NEM316. Survival of the mice following infection was monitored on a daily basis.

[0293] Two independent experiments were performed.

Results

[0294] Maternal vaccination with rGAPDH (whole protein) has previously proven to be an efficient strategy to prevent neonatal infections caused by GBS [24]. However, when antibodies elicited with Neonatal Vaccine were used for passive immunisations of pups before GBS infection, the protection was even more effective. Indeed, the protection conferred with Neonatal Vaccine was 100% (FIG. 17).

Discussion

[0295] This result shows that the new approach used to develop the vaccines of the invention, including Neonatal Vaccine, (i.e. using select peptide sequences, as described herein, instead of the whole protein) directs the immune system of neonates to a more robust and specific response towards sepsis-inducing agents, exemplified by GBS, compared to that previously described.

[0296] This result also shows that the immunity provided by Neonatal Vaccine is reproducible (100% effective), which is clearly advantageous.

[0297] Although this study only looked at GBS infection, it is understood that the same result would be observed upon infection by other sepsis-inducing bacteria (not least because Example 8 shows that antibodies elicited with Neonatal Vaccine recognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and N. meningitidis (as exemplified by MenB)).

[0298] Moreover, the results presented in FIG. 17 provide evidence that maternal vaccination with Neonatal Vaccine (or, therefore, maternal treatment with the anti-GAPDH antibodies of the invention), will significantly reduce stillbirths and premature births caused by intra-vaginal GBS infection. Indeed, as discussed above in connection with the twelfth aspect of the invention, antibodies raised against a peptide, fragment, variant or vaccine of the invention can pass to an unborn baby across the mother's placenta. In addition, and as shown in FIG. 17, the protection against GBS conferred on pups following immunisation with Neonatal Vaccine-induced antibodies was 100%. Owing to the high sequence similarity and functional homology between GAPDH of GBS and the other sepsis-inducing bacteria as described herein, the presented data also indicate that Neonatal Vaccine will effectively prevent stillbirths and pre-term births caused by other sepsis-inducing bacteria too. This is an important finding, as bacterial infections are responsible for approximately 650,000 stillbirths per year worldwide [52,53]. In addition, about 50% of preterm births at less than 32 weeks of gestation are also caused by bacterial infections [9,14,53-56]. The vast majority are caused by maternal commensal bacteria that ascend from the vaginal tract into the amniotic fluid. GBS, E. coli and K. pneumoniae are the most common pathogens found in autopsies of stillbirth babies caused by ascending bacterial infections.

Example 10Neutralisation of Bacterial GAPDH is a Global Approach to Protect Neonates from Bacterial Sepsis

[0299] This study aimed to extend the work described in Example 9, by investigating whether antibody-mediated neutralisation of bacterial GAPDH could prevent neonatal infections caused by the other relevant sepsis-inducing bacteria.

Materials and Methods

[0300] Mice pups were intraperitoneally injected with 80 ?g of Neonatal Vaccine-induced IgG or 80 ?g of control IgG as described above. 12 h later the mice were challenged subcutaneously with 10.sup.7 CFU of S. pneumoniae strain Tigr4, 500 CFU of E. coli strain IHE3034 or 5?10.sup.5 CFU of S. aureus strain NEWMAN. Survival of the mice following infection was monitored on a daily basis.

Results

[0301] As shown in FIG. 18, the use of antibodies elicited with Neonatal Vaccine in passive immunisations of neonates significantly improve survival upon bacterial challenge. Here are shown results for S. pneumoniae, E. coli and S. aureus (see FIG. 18A-C, respectively).

Discussion

[0302] As discussed herein, currently there is no available vaccine directed to any of the most relevant sepsis-inducing bacteria. Presented here are data demonstrating that antibody-mediated neutralisation of bacterial GAPDH prevents neonatal infections caused by the most relevant sepsis-inducing bacteria.

Example 11Therapeutic Administration of Neonatal Vaccine IgG Antibodies Protects Newborn Mice from GBS Infection

[0303] This study aimed to investigate whether antibodies elicited with Neonatal Vaccine could treat an existing neonatal infection caused by sepsis-inducing bacteria.

Materials and Methods

[0304] Neonatal Vaccine IgG, control IgG (80 ?g) or saline solution (0.9% NaCl) were intraperitoneally injected into mice pups (up to 48 h old) 6h after subcutaneous infection with 5?10.sup.6 GBS NEM316 CFU. At the time of treatment all mice presented clear signs of infection, assessed by intense rash at the site of infection. Survival of the mice following infection was monitored on a 12-hourly basis.

Results

[0305] As shown in FIG. 19, only the mice that received Neonatal Vaccine-induced IgG antibodies were able to survive GBS infection. In fact, treatment of mice pups with anti-GAPDH IgG antibodies after GBS infection resulted in complete survival of the animals. In contrast, none of the controls survived the infection.

Discussion

[0306] As discussed herein, the current treatment available for neonatal sepsis is based only on antibiotic administration. Presented here are data demonstrating that antibodies induced by Neonatal Vaccine can be used to treat existing neonatal infections caused by GBS, one of the most relevant sepsis-inducing bacteria.

[0307] Although this study only looked at GBS infection, it is understood that the same result would be observed upon infection by other sepsis-inducing bacteria (not least because Example 8 shows that antibodies elicited with Neonatal Vaccine recognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and N. meningitidis (as exemplified by MenB)).

[0308] The peptides, fragments and variants of the first aspect of the invention thus have significant utility in creating a variety of useful and much-needed antibody-based therapeutics for the indicated patient populations.

Example 12Neonatal Vaccine Protects Old Mice from GBS Infection

[0309] This study aimed to show that the antibodies produced in Example 8 could be used to protect old mice from GBS infection.

Materials and Methods

[0310] Old C57Bl/6 mice (aged over 16 months) were intraperitoneally injected with 1 mg/kg of Neonatal Vaccine-induced IgG or the same amount of isotyped matched IgG as a control daily for three days. 24 h after the last dose the mice were challenged so subcutaneously with 2?10.sup.7 CFU of GBS NEM316. Survival of the mice following infection was monitored on a daily basis for 12 days.

[0311] Two independent experiments were performed.

Results

[0312] Vaccination with Neonatal Vaccine was shown to protect old mice against lethal GBS infection. Indeed, eight out of nine mice (?90%) injected with Neonatal Vaccine survived the bacterial challenge compared to only one of ten (10%) controls (FIG. 20).

Discussion

[0313] This result shows that the vaccines of the invention, including Neonatal Vaccine, can direct the immune system of old mice to a robust and specific response towards sepsis-inducing agents, as exemplified by GBS, in a parallel fashion to that demonstrated in neonates (see Example 9).

[0314] The inventors firmly believe, therefore, that susceptibility to infection by sepsis-inducing bacteria in the elderly is underpinned by the same mechanism as they have discovered in neonates (i.e. GAPDH, which is secreted by the GBS bacteria, acts on B1 cells through TLR2 in order to induce IL-10 production). Indeed, the data provided herein show that Neonatal Vaccine can be used to produce antibodies that recognise bacterial GAPDH produced by GBS, and this is clearly having a protective effect in the old mice, just as has been observed in neonates. The inventors therefore also firmly believe that the same would be true for other such immunocompromised hosts.

[0315] Although this study only looked at GBS infection, it is understood that the same result would be observed upon infection by other sepsis-inducing bacteria (not least because Example 8 shows that antibodies elicited with Neonatal Vaccine recognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and N. meningitidis (as exemplified by MenB)). The results seen with Neonatal Vaccine in neonatal mice challenged with the different bacterial strains (see Example 10) could therefore reasonably be expected in other immunocompromised hosts, such as old mice, too.

[0316] As described herein, currently there is no available vaccine that efficiently protects the elderly against infections caused by any of the most relevant sepsis-inducing bacteria. Therapeutic strategies to combat sepsis in this group are also far from effective. Presented herein are data demonstrating that antibody-mediated neutralisation of bacterial GAPDH prevents infections caused by the most relevant sepsis-inducing bacteria in the elderly. Vaccination is the most cost-effective treatment for infectious diseases, even more so when the same vaccine could prevent infections caused by different human pathogens in different age groups, as has been demonstrated here.

[0317] The data obtained in the old mice are proof-of-concept that the other results obtained in the neonates would be obtained in the elderly and other such immunocompromised hosts too. The administration of Neonatal Vaccine IgG antibodies to old mice suffering an existing infection caused by sepsis-inducing bacteria is therefore expected to result in their treatment, just as has been observed in the neonates (see Example 11). As the current treatment available for sepsis is based only on antibiotic administration, the fact that antibodies induced by Neonatal Vaccine could be used to treat existing infections caused by the most relevant sepsis-inducing bacteria in the elderly, as well as in neonates and the other patient populations indicated herein, is clearly advantageous.

Example 13Neonatal Vaccine Protects NOD Mice Against GBS Infection

[0318] This study aimed to show that the antibodies produced in Example 8 could be used to protect a transgenic mouse model of diabetes (NOD mice) from GBS infection.

Materials and Methods

[0319] NOD mice (eight weeks of age) were intraperitoneally injected with 1 mg/kg of Neonatal Vaccine-induced IgG or the same amount of isotyped matched IgG as a control daily for three days. 24 h after the last dose the mice were challenged subcutaneously with 5?10.sup.7 CFU of GBS NEM316. Survival of the mice following infection was monitored on a daily basis for 12 days.

[0320] Two independent experiments were performed.

Results

[0321] Passive immunisation using Neonatal Vaccine-induced IgG was shown to protect NOD mice against lethal GBS infection. Indeed, seven out of eight mice (?90%) injected with so Neonatal Vaccine-induced IgG survived the bacterial challenge compared to only two of eight (25%) sham-immunised controls (FIG. 21).

Discussion

[0322] This result shows that the vaccines of the invention, including Neonatal Vaccine, can direct the immune system of a transgenic mouse model of diabetes to a robust and specific response towards sepsis-inducing agents, as exemplified by GBS, in a parallel fashion to that demonstrated in neonates (see Example 9) and the elderly (see Example 12).

[0323] The inventors firmly believe, therefore, that susceptibility to infection by sepsis-inducing bacteria in diabetics, as per the elderly, is underpinned by the same mechanism as they have discovered in neonates (i.e. GAPDH, which is secreted by the GBS bacteria, acts on B1 cells through TLR2 in order to induce IL-10 production).

[0324] Indeed, the data provided herein show that Neonatal Vaccine can be used to produce antibodies that recognise bacterial GAPDH produced by GBS, and this is clearly having a protective effect in the diabetic mice, just as has been observed in neonates (and the elderly). The inventors therefore also firmly believe that the same would be true for other such immunocompromised hosts.

[0325] Again, although this study only looked at GBS infection, it is understood that the same result would be observed upon infection by other sepsis-inducing bacteria (not least because Example 8 shows that antibodies elicited with Neonatal Vaccine recognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K. pneumoniae and N. meningitidis (as exemplified by MenB)). The results seen with Neonatal Vaccine in neonatal mice challenged with the different bacterial strains (see Example 10) could therefore reasonably be expected in other immunocompromised hosts, such as diabetics, too.

[0326] As described herein, diabetic patients have increased susceptibility to infection by sepsis-inducing bacteria. Presented herein are data demonstrating that antibody-mediated neutralisation of bacterial GAPDH prevents infections caused by the most relevant sepsis-inducing bacteria in this patient group. As explained, vaccination is the most cost-effective treatment for infectious diseases, even more so when the same vaccine could prevent infections caused by different human pathogens in different age so groups and across different diseases, conditions and disorders, as has been demonstrated here.

[0327] The data obtained in the diabetic mice are also proof-of-concept that the other results obtained in the neonates would be obtained in diabetics and other such immunocompromised hosts too. The administration of Neonatal Vaccine IgG antibodies to diabetic mice suffering an existing infection caused by sepsis-inducing bacteria is therefore expected to result in their treatment, just as has been observed in the neonates (see Example 11).

[0328] As the current treatment available for sepsis is based only on antibiotic administration, the fact that antibodies induced by Neonatal Vaccine could be used to treat existing infections caused by the most relevant sepsis-inducing bacteria in diabetics, as well as in neonates, the elderly and other patient populations indicated herein, is clearly advantageous.

CONCLUDING REMARKS

[0329] The data presented in the Examples show the relevance of vaccines and treatments of the invention, including Neonatal Vaccine, to protect immunocompromised hosts such as neonates, babies, children, women of fertile age, pregnant women, foetuses and the elderly, in particular, from bacterial sepsis.

[0330] Moreover, the rationale of Neonatal Vaccine and the other vaccines and treatments described herein represents significant new inventive steps regarding the previous published results [24,25], namely: [0331] a) The mechanism by which bacterial GAPDH induces IL-10 in the neonatal host; [0332] b) GAPDH-induced IL-10 production is associated with susceptibility to bacterial sepsis caused by different pathogens; [0333] c) GAPDH-induced IL-10 production is a mechanism conserved in human cord-blood cells; [0334] d) GAPDH-induced IL-10 production is a mechanism conserved in leukocytes isolated from the peripheral blood of adult humans; [0335] e) The efficacy of anti-GAPDH antibodies in preventing stillbirths caused by GBS; [0336] f) Antibodies elicited by Neonatal Vaccine (and other vaccines described herein) recognise extracellular GAPDH from GBS, P. aeruginosa, E. coli, S. pneumoniae, K. pneumoniae, S. aureus and N. meningitidis. [0337] g) Neutralisation of bacterial GAPDH by means of passive immunisation with antibodies elicited with Neonatal Vaccine (and other vaccines described herein), protects newborns from sepsis caused by GBS, E. coli, S. pneumoniae and S. aureus; [0338] h) The use of peptides derived from GAPDH of sepsis-inducing bacteria and the use of anti-GAPDH IgG antibodies, either as a preventive strategy or as a treatment for neonatal sepsis and sepsis in other patient groups as indicated herein.

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