CATH2 AND DERIVATIVES FOR INHIBITING STREPTOCOCCUS SUIS

Abstract

The invention relates to methods for inhibiting S. suis comprising administering to CATH2 or a derivative thereof and to methods for the treatment or prevention of a S. suis infection in a subject in need thereof, comprising administering CATH2 or a derivative thereof to the subject.

Claims

1. A method for the treatment or prevention of a Streptococcus suis (S. suis) infection in a subject in need thereof, comprising administering CATH2 or a derivative thereof to the subject.

2. (canceled)

3. (canceled)

4. (canceled)

5. The method according to claim 1 wherein said S. suis is S. suis serotype 2, serotype 9, serotype 1 or serotype 3.

6. The method according to claim 1 wherein said S. suis is serotype 2.

7. The method according to claim 1 wherein the subject in need thereof is suffering from a S. suis infection or at risk of suffering from a S. suis infection.

8. The method according to claim 1 comprising administering said CATH2 or derivative thereof to subjects of a population of subjects wherein a S. suis infection has been established in one or more subjects of said population.

9. The method according to claim 1 wherein the subject is administered the CATH2 derivative twice.

10. The method according to claim 9 wherein the subject is administered the CATH2 derivative with an interval of at least 2 days.

11. The method according to claim 1 wherein the subject is poultry and the administration is performed in ovo and/or after hatch.

12. The method according to claim 1 comprising inducing or promoting innate immune memory in the subject.

13. The method according to claim 1 comprising improving or enhancing antimicrobial treatment with an antimicrobial agent.

14. A method for inhibiting Streptococcus suis comprising administering CATH2 or a derivative thereof to the S. suis.

15. (canceled)

16. The method according to claim 1 wherein the CATH2 derivative is selected from the group consisting of DCATH2, a C-terminally and/or N-terminally truncated CATH2 and a C-terminally or N-terminally truncated DCATH2.

17. The method according to claim 16, wherein the CATH2 derivative is selected from the group consisting of DCATH2, DCATH2(1-21), DCATH2(4-21), CMAP4-21, CMAP5-21, CMAP6-21, CMAP7-21, CMAP8-21, CMAP9-21, CMAP10-21, CMAP11-21, CMAP4-21 (F5.fwdarw.W), CMAP4-21 (F5.fwdarw.Y), CMAP4-21 (F12.fwdarw.W), CMAP4-21 (F12.fwdarw.Y), CMAP4-21 (F5, F12.fwdarw.W), CMAP4-21 (F5, F12.fwdarw.Y), CMAP4-21 (F5.fwdarw.W, F12.fwdarw.Y), CMAP4-21 (F5.fwdarw.Y, F12.fwdarw.W), CMAP7-21 (F12.fwdarw.W), CMAP7-21 (F12.fwdarw.Y), CMAP10-21 (F12.fwdarw.W) and CMAP10-21 (F12.fwdarw.Y).

18. The method according to claim 16, wherein the CATH2 or derivative is DCATH2, DCATH2(1-21) or DCATH2(4-21).

19. The method according to claim 1 wherein the CATH2 or a derivative thereof is combined with an adjuvant specific for innate immunity.

20. The method according to claim 1 wherein the CATH2 or derivative thereof is administered before, after or simultaneously with a treatment with a S. suis or an antigenic part thereof.

21. The method according to claim 7 wherein the subject suffering from a S. suis infection or at risk of suffering from a S. suis infection is a subject that is in contact with subjects suffering from said infection.

22. The method according to claim 19 wherein the adjuvant specific for innate immunity is selected from the group consisting of a toll-like receptor (TLR) ligand, ?-glucan, muramyl dipeptide (MDP), Bacille Calmette-Guerin (BCG), CpG oligodeoxynucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1: .sub.D-CATH2 and its derivatives efficiently kills several S. suis type2 strains in both THB and RPMI+FCS. Antibacterial activity of .sub.D-CATH2 and its derivatives against 10.sup.6 CFU ml.sup.?1 S. suis type 2 strains (P1/7, D282, 5735, and OV625) was tested using a colony count assay in both THB medium (A) and RPMI+FCS (B). 2 log CFU mL.sup.?1 was set as detection limit for the experiment. Data is plotted as average +/?SEM (N=3-4).

[0074] FIG. 2: Peptide titration on BMDM and BMDCs for cytotoxicity and expression of cell markers. Mouse BMDM (A, C, E, G) and BMDCs (B, D, F, H) were cultured for 6 days with GM-CSF and M-CSF respectively. Different concentrations were added at day 6 (A, B, E, F) or the cells were primed with different concentrations peptides at day 1-2 (C, D, G, H). At day 7, cell viability was tested using WST-1 reagent, with no-peptide-control set to 100% viability (A-D) and cells were analyzed by flowcytometry for cell marker expression (E-H). Results are depicted as mean +/?S.E.M. (n=3).

[0075] FIG. 3: .sub.D-CATH2 and its derivatives inhibit LTA-SA- or S. suis-induced activation. Mouse BMDM cells were cultured for 6 days before they were activated with different S. suis type2 strains at an MOI of 0.2. Bacteria were mixed for 5 minutes with 1.25 ?M .sub.D-CATH2 or its derivatives before stimulation. After 24 hours of stimulation, cells were analyzed by flowcytometry (A) and cytokine expression was measured (B). 5*10.sup.5 splenocytes, freshly isolated from WT mice using a digestion buffer followed by filtering through a 40 ?M cell filter, were activated with different S. suis type2 strains premixed with 5 ?M .sub.D-CATH2 or its derivatives at an MOI of 0.2. After 24 hours of stimulation, secreted cytokines were measured using ELISA (C). Data is plotted as average +/?SEM (N=3-6).

[0076] FIG. 4: .sub.D-CATH2 and its derivatives bind to LTA.

Thermodynamic binding capacity of 200 ?M .sub.D-CATH2 (A), .sub.DC(1-21) (B), and .sub.DC(4-21) (C) to 200 ?M LTA-SA was measured using isothermal titration calorimetry (ITC). Every 300 seconds, 1.99 ?l peptide solution was titrated into 164 ?l LTA solution. The corrected heat rate (?J sec.sup.?1) is plotted (top panel) and normalized integrated heat was plotted against the molar ratio between LTA and the peptide (lower panel). Experiments (N=3) were averaged before plotting and fitting an independent model. The corrected heat rate of .sub.D-CATH2, .sub.DC(1-21), and .sub.DC(4-21) is depicted for comparison (D).

[0077] FIG. 5: .sub.D-CATH2 and its derivatives increase the BMDM efficiency.

Mouse BMDM cells were cultured for 6 days. At day 1, 1.25 ?M .sub.D-CATH2 or its derivatives were added for 24 hours. At day 6 the cells were activated with different S. suis type2 strains at an MOI of 0.2. Bacteria were mixed for 5 minutes with 1.25 ?M .sub.D-CATH2 or its derivatives before stimulation. After 24 hours of stimulation, cells were analyzed by flowcytometry (A) and cytokine expression was measured (B). Data is plotted as average +/?SEM (N=3-6).

[0078] FIG. 6: Dendritic cell primed by peptides have increased macrophage markers. Mouse BMDCs were cultured for 6 days with M-CSF and either primed with 1.25 ?M peptides at day 1-2 (A) or 1.25 ?M peptide was added at day 6 during stimulation (B). Cells were analyzed by flowcytometry for cell marker expression. Results are depicted as mean +/?S.E.M. (n=3).

[0079] FIG. 7: Prophylactic .sub.DC(1-21) s.c. injection reduces the clinical symptoms of S. suis P1/7 in mice.

A schematic overview of the in vivo experiment set up. At day 1, all mice were subcutaneously injected with .sub.DC(1-21) or a control in the neck region. Either after 24 hours (24 h .sub.DC(1-21) or 7 days (7 d .sub.DC(1-21) the mice were intra-peritoneally injected with 10.sup.7 CFU S. suis P1/7 or only THB. 24 hours after infection, a few drops of blood were collected and 7 days post infection, the mice were sacrificed for analysis. The black arrows indicate the moment of animal welfare evaluation by weighing and score for clinical symptoms (A). The relative weight difference with the weight at the moment of infection is set to 100% is depicted for 24 h .sub.DC(1-21) (B) and 7 d .sub.DC(1-21) (E). The cumulative clinical score of 8 different parameters is depicted for 24 h .sub.DC(1-21) (C) and 7 d .sub.DC(1-21) (F). Survival curves are depicted and bacterial counts in different organs of mice reaching HEP is depicted for 24 h .sub.DC(1-21) (D) and 7 d .sub.DC(1-21) (G). The number of organs per mouse in which S. suis bacteria were found (H) and the average CFU per organ per mouse (I). The bacterial burden of mice died before the end of the study with circles depicting mice infected 24 hours post peptide injection and squares 7 days post peptide injection (J). Results are depicted as mean +/?S.E.M. (CNTR n=4, CNTR+S.suis n=12, .sub.DC(1-21) n=4, and .sub.DC(1-21)+S. suis n=12).

[0080] FIG. 8: Bacterial counts in the blood and different organs of S. suis infected mice. 24 hours post infection, blood was drawn via cheek puncture (A) and via heart puncture after 7 days (B) and plated on TSA/5% sheep blood plates for bacterial counts (A,B). The peritoneum was flushed at day 7 and cells present in the peritoneal lavage (PTL) were counted (C). The spleens were weighed (D). The single cell suspension of the different organs was plated on TSA/5% sheep blood plates for bacterial counts. CFU per mg organs was calculated (E).

EXAMPLES

Materials and Methods

Peptides, Bacterial Strains and Experimental Animals

[0081] The 26 amino acid full .sub.D-antiomer of chicken CATH2 (RFGRFLRKIRRFRPKVTITIQGSARF-NH.sub.2) (.sub.D-CATH2) with a net positive charge of 9 (9+) and two derivatives (.sub.DC(1-21), 8+ and .sub.D(4-21), 7+) were used in this study. The peptides were synthesized by Fmoc-chemistry at China Peptides (CPC scientific, Sunnyvale, CA, USA) and purified by reverse phase high-performance liquid chromatography to a purity of >95%. Lyophilized peptides were dissolved in endotoxin free water.

S. suis Serotype 2 strain P1/7, D282, 5735, and OV625 were used in this study. All strains have been previously characterized.[17] Bacterial strains were grown overnight from glycerol stocks in Todd-Hewitt broth (THB) (Oxoid Ltd., London, UK) before use.
Seven- to ten-week-old Crl:CD-1 mice (both male and female) were purchased from Charles River. All mice were kept under specific pathogen-free conditions with free access to food and water under the guidelines for animal experimentation as approved by the Dutch central authority for scientific procedures on animals (CCD).

Antibacterial Activity

[0082] S. suis Serotype 2 strains P1/7, D282, S735, and OV625 were grown into mid-logarithmic phase for 3-4 hours at 37? C. in THB, after which bacteria were centrifuged at 1200?g for 10 minutes at 4? C. and resuspended in fresh THB. Concentration was determined by measuring the OD value at 620 nm with an OD of 0.1 is 1?10.sup.8 colony forming units (CFU) mL.sup.?1. 10.sup.6 CFU mL.sup.?1 S suis was mixed with different concentrations of .sub.D-CATH and derivatives (0.63?40 ?M) and left for 3 hours at 37? C. Ten-fold dilutions were prepared and spread in Tryptan Soy agar (TSA) plates containing 5% (vol/vol) defibrinated sheep blood (Oxoid) and colonies were allowed to grow for 48 hours. Minimal Bactericidal Concentration (MBC) was defined as ?100 CFU mL.sup.?1 (2 logCFU mL.sup.?1), the detection limit of the assay.

Cell Culture and Flow Cytometry

[0083] Bone marrow cells, isolated from the femur and tibia of both hindlegs, were stored in FCS/10% DMSO in liquid nitrogen. Cells were grown at a concentration of 5?10.sup.5 cells mL.sup.?1 in RPMI-1640 without phenol red (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal calf serum (FCS) (Corning, NY, USA) and 1% Penicillin/streptomycin (Thermo Fisher Scientific). Bone marrow derived macrophages (BMDM) and bone marrow derived dendritic cells (BMDC) were culture by adding 20 ng mL.sup.?1 murine recombinant M-CSF or GM-CSF (Peprotech, NJ, USA) respectively. If indicated, cells were trained by adding 1.25 ?M peptide at day 1, which was replace by fresh medium at day 2. The medium of all cells was replaced by fresh medium without antibiotics at day 3. At day 6 cells were stimulated with 1 ?g mL.sup.?1 lipoteichoic acid from S. aureus (LTA-SA) (Invivogen) or with the different S suis strains with a multiplicity of infection (MOI) of 0.2. Medium containing S. suis was removed after 2 hours and replaced by medium containing 200 ?g/ml gentamycin (Sigma-Aldrich, MO, USA) and left for an additional 22 hours. After 24 hours, medium was collected and stored at ?20? C. for cytokine measurements. Cells were incubated for 5 min with PBS/0.5 mM EDTA after which they were resuspended by vigorous pipetting and used for flow cytometry. Cells were resuspended in flow cytometry buffer (PBS/0.5% BSA (Sigma Aldrich)) and kept on ice during the whole procedure. Cells were stained with antibodies (table 1) for 20 minutes, washed and measured using the BD FACSCanto-II (BD bioscience) and analyzed with FlowJo software (Ashland, OR, USA).

Splenocytes Activation

[0084] Mice were killed using CO.sub.2 suffocation after which the spleen were harvested. Spleen were digested with digestion buffer (1.5 WU/ml liberase TL grade (Roche, Basel, Switzerland), 100 Units/ml recombinant DNAse I (Roche) for 30 minutes at 37? C. and meshed through a 40 ?m filter (BD bioscience) to prepare single cell solution using PBS/0.5 mM EDTA wash buffer. The red blood cells were lysed using an isotonic ammonium chloride buffer (155 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA) for 5-10 minutes on ice, washed 1? with PBS, after which the cells were counted and resuspended in in high glucose DMEM (Thermo Fisher scientific, MA, USA) supplemented with 10% FCS (Corning, VA, USA). 5?10.sup.5 splenocytes were added per well in a U-bottom 96-wells plate. Total splenocytes were stimulated with 1 ?g LTA-SA or the different S. suis strains at an MOI of 0.2. After 2 hours, the supernatant was collected (by centrifugation 1800 RPM, 2 min) and the cells were resuspended in 100 ?l fresh medium supplemented with 200 ?g/ml gentamycin and left for an additional 22 hours. After 24 hours, medium was collected and stored at ?20? C. for cytokine measurements.

Cell Viability and Activity

[0085] WST-1 reagent (roche) was used for cell viability of BMDCs and BMDM as well as for cell activity of activated splenocytes. In both cases, 100 ?l fresh medium containing 10% WST-1 was added and incubated at 37? C. After 30-60 minutes, colorimetric changes were measured at 450 nm using a FLUOstar Omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The metabolic activity is depicted as a percentage with the untreated BMDCs/BMDMs or unstimulated splenocytes set to 100%.

ELISA

[0086] TNF?, IFN?, IL-1?, and IL-6 were measured in the supernatant (diluted in PBS/5% BSA if needed) using a Duoset ELISA kit (R&D systems, MN, USA). ELISAs were performed according manufacturer's instructions. Colorimetric changes were measured at 450 nm using a FLUOstar Omega microplate reader (BMG Labtech GmbH).

Isothermal Calorimetry (ITC)

[0087] The interaction between the .sub.D-CATH2 peptides and LTA-SA was tested using isothermal titration calorimetry (ITC). All ITC experiments were performed on a Low Volume NanoITC (TA instruments-Waters LLC, New Castle, USA). 800 ?M of LTA-SA or peptide solution was prepared in MilliQ after which a 4-fold dilution in dPBS (Gibco) was made. The chamber was filled with 164 ?l LTA-SA and the peptide was loaded in the syringe. Every 300 seconds, 1.99 ?L peptide was titrated into the chamber at 37? C. Data was analyzed using the Nano Analyze software (TA instruments-Waters LLC). The data of three experiments was averaged and an independent model was used to determine the peptide-LTA interaction.

In Vivo Infection Experiment

[0088] Upon arrival, mice were allowed to acclimate for at least 7 days before the start of the experiment. The experiment was performed as depicted in FIG. 7A. The experiment was repeated twice to obtain in total 4 mice in the control groups and 12 mice in the infection groups. At day 1, mice were s.c. injected in the neck region with 1 mg kg.sup.?1 .sub.DC(1-21) in PBS/Cholesterol or with PBS/cholesterol alone. The peptide and control groups were blinded to avoid any influence by the researchers. After 24 hours (group 1) or after 7 days (group 2), mice were i.p. infected with 10.sup.7 CFU S. suis P1/7 in THB or with THB alone as control. 24 hours after infection, a few drops of blood were collected via cheek puncture for bacterial count. During the infection phase of the experiment, mice were checked every 12 hours in the acute phase of disease (first 48 hours) and thereafter daily until the end of the end of the study. A cumulative clinical score was given to the mice as measure of disease using several parameters as depicted in table 4, according to Seitz et al.[18] When a mouse obtained a clinical score of 2 on 3 of the 8 points 2 days in a row or in case of severe weight loss (>20%), the mouse was euthanized for animal welfare reasons (humanized end point (HEP)) and the organs were collected for bacterial counts as described hereafter. 7 days post infection all mice were sacrificed for further analysis. Mice were anesthetized using Isoflurane and 1 mL blood was drawn via heart puncture, followed by cervical dislocation. The peritoneum was flushed with 5 mL PBS/0.5 mM EDTA and diluted in 10 mL ice cold PBS/0.5% FCS. The organs (spleen, lungs, liver, lymph nodes (axillary, inguinal, and mesenteric), brain, kidney and bone marrow) were collected and stored in ice cold PBS. All organs, except the bone marrow and lymph nodes were weight using a sartorius microbalance. The peritoneal lavage (PTL) samples were counted using the Countess II Automated Cell Counter (Thermofisher). The lungs, liver, brain and kidney were meshed through a 40 ?m filter (BD bioscience) with 5 mL PBS to obtain single cell suspensions. Spleen and lymph nodes were digested with digestion buffer (1.5 WU/ml liberase TL grade (Roche, Basel, Switzerland), 100 Units/ml recombinant DNAse I (Roche) for 30 minutes at 37? C. and meshed through a 40 ?m filter using 5 mL PBS/0.5 mM EDTA. The red blood cells of the blood and spleen were lysed using an isotonic ammonium chloride buffer (155 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA) for 5-10 minutes on ice, washed 1? with PBS and were resuspended in FACS buffer (PBS/0.5% BSA). Bone marrow samples were flushed out the femur and tibia of both legs with 5 mL PBS and filtered through a 40 ?m filter. A sample of the blood, bone marrow, spleen, peritoneal lavage and lymph nodes was taken and stained for 30 minutes with different antibody panels (table 1) and measured using the BD FACSCanto-II and analyzed with FlowJo software. Of the lungs, liver, brain, kidney, spleen and peritoneal lavage samples, a 10-fold serial dilution was prepared and the samples were plated on TSA plates containing 5% (vol/vol) defibrinated sheep blood. The colonies were allowed to grow for 48 hours. The number of colonies were counted, with ?100 CFU mL-1 (2 logCFU mL-1) as detection limit of the assay, and calculated as CFU/mg organ.

TABLE-US-00004 TABLE 1 Antibodies used Antigen Clone Label Manufacture MHC-II M5/114.15.2 FITC eBioscience CD11c HL3 PE BD Bioscience Sirp-? P84 PerCP- eBioscience eFluor710 CD19 1D3 PE-Cy7 BD Bioscience CD8? 53-6.7 APC BD Bioscience CD11b M1/70 APC-Cy7 BD Bioscience CD24 M1/69 eFluor450 eBioscience CD86 GL-1 PerCP BoiLegend F4/80 BM8 APC eBioscience Ly6C HK1.4 eFluor450 eBioscience CD4 RM4-5 AF488 BD Pharmingen CD62L MEL-14 PE eBioscience CD335 29A1.4 PerCP-Cy5.5 BD Bioscience CD44 IM7 PE-Cy7 BD Bioscience CD3e 145-2C11 APC-Cy7 BD Pharmingen CD25 eBio3C7 eFluor450 eBioscience Antibodies used for flow cytometry. All antibodies were diluted 1000x in flow cytometry buffer prior to use (CD19 and CD335 were used in a 500x dilution).

Statistics

[0089] Samples were compared to no-peptide-controls using two-way ANOVA with the Dunnett post-hoc test. Samples were paired for cell culture samples. *=p?0.05; **=p?0.01; ***=p?0.001;****=p?0.0001.

Results

.SUB.D.-CATH2 and its Derivatives Efficiently Kills Several S. Suis Type2 Sub-Strains in Both THB and RPMI+FCS

[0090] Antimicrobial activity of d-CATH2 and its derived peptides was assessed against 4 different S. suis serotype 2 strains. The mean bactericidal concentration (MBC) of the three peptides is 2.5-5 ?M for the four sub-strains in bacterial growth medium THB (FIG. 1A and Table 2). However, most of the assays will be performed in cell culture medium RPMI+10% FCS and it has been shown that medium can influences the activity of cathelicidins,[19,20] the MBC of .sub.D-CATH2 and its derived peptides was also tested in RPMI+10% FCS medium. The activity of .sub.D-CATH2 and .sub.DC(1-21) slightly increased to 0.6-2.5 ?M, whereas the shortest peptide, .sub.DC(4-21), remained with a MBC of 2.5 ?M (FIG. 1B and Table 2), indicating that either charge or number of amino acids is important for the antibacterial function of .sub.D-CATH2.

TABLE-US-00005 TABLE 2 MBC values of D-CATH2 killing S. suis strains THB RPMI + 10% FCS D- DC DC D- DC DC CATH2 (1-21) (4-21) CATH2 (1-21) (4-21) P1/7 2.5-5 2.5 5-10 1.25-2.5 1.25 2.5 S735 1.25-2.5 2.5 2.5 0.6-2.5 1.25 2.5 D282 2.5-5 2.5-5 2.5-5 0.6-2.5 0.6-1.25 1.25-2.5 OV625 1.25-5 0.6-5 1.25-2.5 1.25-2.5 1.25 2.5 MBC values for the different peptides depending on the bacterial strain and the medium.

.SUB.D.-CATH2 and its Derivatives Inhibit LTA-SA- or S. Suis-Induced Activation by Binding to LTA

[0091] The biological form of CATH2 is known to inhibit LPS and LTA activation of a murine macrophage cell-line,[21] however, whether the full D antiomer of CATH2 is also capable of inhibiting LTA-induced of primary cultured murine BMDMs and BMDCs activation is unclear. In addition, cathelicidins can be cytotoxic to mammalian cells in higher concentrations.[19] Therefore, murine BMDMs and BMDCs were exposed to .sub.D-CATH2, .sub.DC(1-21) and .sub.DC(4-21), either added at the end of the culture (day 6) or at the beginning of the culture (day 1) to observe any effects of the peptides on the cell viability and differentiation.
BMDMs were relative sensitive to addition of .sub.D-CATH2 and its derivates, especially to .sub.DC(1-21) (FIG. 2A), whereas BMDCs had some reduced viability, but starting from 2.5 ?M peptides with no difference between the three (FIG. 2B). Both BMDM and BMDCs were less sensitive if the peptides were added at the beginning of the culture, with only a small reduction in viability at 5 ?M (FIG. 2C and D). Analysis with flow cytometry also show a decrease in % BMDMs. The surviving BMDMs have an increased F4/80 expression and reduced MHC-II (FIG. 2E). A minor reduction in BMDCs is only visible at 5 ?M, without affecting the other cell markers (FIG. 2F).
To analyze the effect of stimulation in combination with peptides, 1.25 ?M was chosen as concentration on the border of cytotoxicity, but still influencing the expression of F4/80 with macrophages. Four different sub-strains of S. suis serotype 2 were mixed with 1.25 ?M peptide and added to BMDMs at day 6. Activation of BMDMs in combination with the peptides, did not influence the percentage of macrophages as shown by flow cytometry. However, the upregulation of activation markers, like MHC-II, CD86 and CD38, was strongly inhibited by all three peptides for all four sub-strains (FIG. 1A). Similar results were found for BMDCs (FIG. 6A). In addition, the secretion of TNFa and IL-6 was inhibited in the presence of the peptides (FIG. 3B). To study the influence of the peptides on S. suis-induced activation in a more complex system, total splenocytes were activated ex vivo. In addition to whole live S. suis bacteria, pure LTA was used for activation. In the presence of peptides, nor LTA or whole S. suis bacteria were able to activate the splenocytes, shown by the inhibition of TNF? and IL-6 secretion (FIG. 3C).
To study whether to inhibitory effect on activation by the peptides to a direct interaction with LTA, the LTA binding capacity of peptides was tested using isothermal titration calorimetry (ITC). Although LTA- and S. suis-induced activation was strongly inhibited by all three peptides, is partially explained by its direct binding to LTA. With a dissociation coefficient K.sub.d between 2-10 ?M. Interestingly, .sub.DC(1-21) bind less strong compared to the other two peptides, with a lower K.sub.d and less peptide binding to one LTA molecule (FIG. 4 and Table 3), although the three peptides are equally efficient in inhibiting LTA- and S. suis-induced activation.

TABLE-US-00006 TABLE 3 ITC data D-CATH2 DC(1-21) DC(4-21) K.sub.d (?M) 3.039 10.22 2.12 n 0.543 0.207 1.209 ?H ?21.16 ?85.64 ?16.73 (kJ/mol) ?S 37.39 ?180.6 54.68 (J/mol .Math. K)
Overview of ITC results of the binding capacity of 200 ?M .sub.D-CATH2, .sub.DC(1-21) or .sub.D-C(4-21) to 37.2 ?M LTA-SA. K.sub.ddissociation coefficient (?M); nnumber of peptide molecules binding to one LPS molecule; ?Henthalpy changes; -?Sentropy changes.
.sub.D-CATH2 and its Derivatives Increases the BMDM Culture Efficiency To further study the effect of .sub.D-CATH2 and its derivatives on macrophages, cells were exposed to the peptides 24 hours after the start of the culture for 24 hours. The efficiency of the BMDMs was enhanced by the early exposure of the peptides, shown by a higher percentage macrophages at day 6, which was most pronounced for .sub.DC(1-21) (FIG. 5A). However, priming of the cells did not influence the activity of the cells for it did not change the activation markers MHC-II, CD86 and CD38 (FIG. 5A), nor was there any difference in cytokine expression by the peptide treated cells (FIG. 5B).
Similar results were found in the BMDC culture, exposing the cells to the peptides 24 hours after the start of the culture for 24 hours. Although the percentage of BMDCs at day 6 did not change, nor was there any difference in the expression of the activation markers, the macrophage marker F4/80 was increased, indicating a skewing towards macrophage like cells (FIG. 6B).

.SUB.D.C(1-21) Reduces the Clinical Symptoms of S. Suis P1/7 in Mice

[0092] Previously, our group has shown that in ovo injection of .sub.D-CATH2 three days before hatch, protects the chickens up to 7 days post hatch for infection.[22] Since addition of .sub.D-CATH2 and more specifically .sub.DC(1-21) enhanced the efficiency of the murine BMDM culture and balanced the inflammatory response, we questioned whether injection of .sub.DC(1-21) could boost the immune response in mice as well. Therefore, mice were injected with 1 mg/kg .sub.DC(1-21) at day 1 subcutaneously and infected with 10.sup.7 CFU/ml S. suis P1/7 intraperitoneally 24 hours or 7 days post peptide injection. Mice were weighed twice a day during the acute phase of infection and daily until 7 days post infection (FIG. 7A). Both peptide-treated mice as control mice lost around 8% bodyweight up to 48 hours post infection and started to gain weight again due to S. suis infection, independent whether infection was started 24 hours (FIG. 7B) or 7 days post peptide injection (FIG. 7E). In addition, a cumulative clinical score was given twice a day during the acute phase of infection and daily during the chronic phase to the mice using a scoring table (Table 4). This shows a small reduction of cumulative clinical score for mice in the late stage of disease if mice were infected 24 hours post peptide injection (FIG. 7C) or at the acute phase of disease if infected 7 days post peptide injection (FIG. 7F). In addition to the reduced cumulative clinical score, treated mice had a higher change of survival when infection was given 24 hours (FIG. 7D) or 7 days post peptide injection (FIG. 7G).

[0093] Bacterial counts in the different organs were determined as well. 24 hours post infection, all mice, treated or not, had S. suis bacteria in the bloodstream between 10.sup.5-10.sup.6 CFU mL.sup.?1 (FIG. 8A). After 7 days, most mice were able to deplete all bacteria in the blood, which was more efficient in 24 h .sub.DC(1-21) treated mice compare to untreated (FIG. 8B). During the section at the end of the study, the peritoneum was washed and the cells present in the peritoneal lavage were counted, showing an increase upon infection, but not different for treated or untreated mice (FIG. 8C), nor were differences found with flow cytometry (data not shown), lastly, the amount of bacteria were determined showing that most mice were able to clear the bacteria in the peritoneum at similar rates in the treated and untreated groups (FIG. 8E). The spleens of S. suis infected mice were enlarged, showing an efficient infection model, however, no differences were found between treated and untreated mice (FIG. 8D). The amount of S. suis in the different organs was determined (FIG. 8E), only minor differences were found. However, counting the number of organs in which bacteria could be found, showed that 24 h .sub.DC(1-21) treated mice had less positive organs (FIG. 7H) and less total CFU counts (FIG. 7I) compare to the untreated mice. This effect was not found for 7d .sub.DC(1-21) treated mice. Also .sub.DC(1-21) treated mice reaching the HEP before the end of the study, showed less bacterial counts, especially in the brain indicating a less severe course of disease(FIG. 7J). The immune cells were analyzed for the different organs; however, no differences were found between treated and untreated mice.

TABLE-US-00007 TABLE 4 Clinical scoring parameters for cumulative scoring of S. suis-infected mice SCORE 0 1 2 BODY WEIGHT Constant or gain >5% weight >20% weight loss loss COAT Flat and glossy Rougher Bloated BREATHING Rhythmic Rapid Rapid and abdominal DEHYDRATION Normal skin Reduced skin Persisting skin elasticity elasticity fold BEARING Normal Curved back Huddled EYES Normal Moderately Squeezed and squeezed swollen ACTIVITY Normal Reduced activity Apathy LOCOMOTION Normal Reduced Unsteady, coordination apraxia

[0094] The cumulative clinical score was defined as the sum of the clinical scoring for eight parameters. Mice were euthanized for animal welfare reasons (humanized end point (HEP)) when they endured severe clinical signs (defined as: 2 days in a row a score of 2 on 3 of the 8 points) or in case of severe weight loss (>20%).

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