Bactericidal/permeability increasing protein for use in a method of immunization, preferably as an adjuvant in a method of vaccination

Abstract

The present invention relates to bactericidal/permeability increasing protein (BPI) for use in a method of immunization of a patient, preferably as an adjuvant in a method of vaccination. The present invention also relates to a preparation comprising BPI for use in a method of immunization of a patient, and optionally an immunomodulatory agent. The present invention further relates to a process of producing a preparation including BPI for use in a method of immunization of a patient.

Claims

1. A method of immunization of a patient, said method comprising administering to the patient an effective amount of bactericidal/permeability-increasing Protein (BPI)-stimulated target cells, wherein said method comprises administering BPI as a stimulant to target cells in vitro or ex vivo, wherein said stimulated target cells are subsequently administered to said patient in vivo, wherein the target cells are dendritic cells, wherein BPI stimulates the dendritic cells to produce cytokines that promote lymphoid cell responses, wherein said immunization prevents or treats a disease selected from chronic inflammatory diseases and graft-versus-host disease (GvHD), and/or wherein said immunization treats a cancerous disease.

2. The method according to claim 1, wherein BPI is coadministered with an immunomodulatory agent selected from the group consisting of BPI-ligands, check-point inhibitors, TLR ligands, CLR ligands, CD1 ligands, inflammasome activators, alarmines and other pathogen-associated or danger-associated molecular patterns, lipoarabinomannans, phosphatidyl inositol mannosides, lipomannans, phospholipids, compounds containing a diacyl-thioglycerol motif, immunostimulatory agents displaying an acyl-anchor, antibodies, cytokines, chemokines, nucleic acids, and nucleic acid analogs.

3. The method according to claim 2, wherein said immunomodulatory agent is an immunostimulatory agent, wherein said immunostimulatory agent is a bacterial lipopeptide (bLP) or-protein, a lipoteichoic acid or CpG ODN, or is a CLR ligand.

4. The method according to claim 2, wherein said immunomodulatory agent is an immunostimulatory agent selected from lipoarabinomannans, nucleic acids, nucleic acid analogs, and immunostimulatory agents displaying an acyl-anchor.

5. The method according to claim 1, wherein BPI is coadministered with an antigen.

6. The method according to claim 5, wherein said antigen is covalently coupled to BPI and/or to a BPI-ligand and/or to a carrier, or wherein said antigen is coadministered with said BPI, but not covalently coupled therewith, using a joint pharmaceutically acceptable delivery system for said antigen and said BPI, or wherein said antigen is coadministered with said BPI, but not covalently coupled therewith, using a separate pharmaceutically acceptable delivery system for each of said antigen and said BPI.

7. The method according to claim 5, wherein BPI is administered as a BPI-encoding nucleic acid within a vector, and wherein a nucleic acid sequence of said antigen is encoded by the same vector as said nucleic acid sequence of BPI, or wherein a nucleic acid sequence of said antigen and said nucleic acid sequence of said BPI are each encoded by a separate vector, or wherein BPI is administered as a peptide and said antigen is administered being encoded by a vector, or wherein BPI is administered being encoded by a vector and said antigen is administered as a peptide.

8. The method according to claim 1, wherein said in vitro or ex vivo stimulated target cells trigger generation of specific T cells, or generation of innate lymphoid cells, NK cells.

9. The method according to claim 8, wherein the method triggers the generation of T cells selected from Th1 cells, Th17 cells, Th22 cells, Tfh cells, regulatory T cells, yo T cells, and cytotoxic T cells.

10. The method according to claim 1, wherein the lymphoid cell responses are Th1, Th17, Th22, regulatory T cell and/or cytotoxic T cell immune responses.

11. The method according to claim 1, wherein said immunization treats chronic inflammatory diseases.

12. The method according to claim 1, wherein said immunization treats GvHD.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) In the following, reference is made to the figures:

(2) All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

(3) FIG. 1 shows FACS analysis data showing surface expression of maturation markers/costimulatory molecules, which are required for T cell activation by bone marrow derived dendritic cells (BMDCs). When BPI is used to stimulate said cells, it upregulates these maturation markers/costimulatory molecules in such stimulated cells compared to unstimulated cells (n=4).

(4) FIG. 2 shows the influence of BPI on dendritic cells (DCs). Human and murine BPI induce comparable genes in bone-marrow derived dendritic cells indicating no species barrier. When BPI is used to stimulate said cells, it induces expression of IL-2, IL-12p40, IL-6, CXCL10, and others, in such stimulated cells compared to unstimulated cells. This indicates that BPI promotes secretion of IL-2, IL-12, and CXCL10, and thus Th1 cell differentiation, and IL6 to promote Th17, Th22 and Tfh differentiation. A. Gene expression in murine DCs stimulated with murine and human BPI (microarray, 4h). A linear relation between huBPI and muBPI was observed indicating that no species barrier exists. B. Examples of induced genes with importance for activation and priming of T cells and other lymphoid cells, such as IL-1b, IL-2, IL-6, IL-10, IL-12b, IL-15, CXCL10, TNF, CD40 and CD86, and chemokines important for the attraction of immune cells, such as MCP-1, CXCL9, CXCL10 and CXCL11 (microarray, 4h). Importantly, IL-2 is the highest induced cytokine. C. Example for gene induction by BPI as shown for IL-2, CXCL10, IL-12p40, and IL-6 in murine DCs using rtPCR.

(5) FIG. 3 shows expression of IL-2, IL-12p40, and IL-6 after stimulation with BPI, Zymosan depleted (Zymd), and a bacterial lipopeptide (bLP, (R)-Pam3CSK4). BPI is a superior stimulus especially towards IL-2 secretion as compared to adjuvants like TLR and CLR ligands. Importantly, Zymd is known for its high potency to induce of IL-2 in BMDCs. However, BPI is a more potent inductor of IL-2. The stimulatory effect of BPI on the expression of IL-2 and IL-12p40/p70 in murine DCs significantly exceeds the stimulatory effect of Zymd and bLP, whereas the stimulatory effect of BPI, Zymd, and bLP on the expression of IL-6 is similar, as observed using Luminex (n>7, 4h, meanstandard error of the mean, ratio paired student's t test).

(6) FIG. 4 shows expression of TNF in murine dendritic cells (BMDC) and murine macrophages (BMDM) upon stimulation with BPI and LPS Ec. BPI preferentially stimulates cells that are subtypes of immune cells, particularly subtypes of antigen-presenting cells, such as dendritic cells as shown for BMDCs. No induction of TNF by BPI is seen in BMDMs. A. DCs react towards both stimuli with secretion of TNF (n>3, 18h, meanstandard error of the mean, paired student's t test). B. Macrophages respond to LPS Ec but not to BPI as observed by ELISA (n>3, 18h, meanstandard error of the mean, paired student's t test).

(7) FIG. 5 shows experimental data addressing the synergism of BPI and bacterial lipopeptides (bLPs) in stimulation of immune cells. A synergistic immunostimulatory effect was observed for BPI in combination with bLP in human peripheral blood mononuclear cells. A. Binding of bLP by BPI as exemplified for (R)-Pam3CSK4 (microscale thermophoresis, MST). B. Synergism of BPI and bLP in the stimulation of human peripheral blood mononuclear cells (TNF ELISA, n=7, 18h, meanstandard error of the mean, paired student's t test). C. Binding of LTA SP by BPI (microscale thermophoresis, MST), but no binding of WTA SP. D. Synergism of BPI and LTA SP in the stimulation of human peripheral blood mononuclear cells (TNF ELISA, n=4, 18h, meanstandard error of the mean, paired student's t test).

(8) FIG. 6 shows experimental data addressing the synergism of BPI and bacterial lipopeptides (bLPs) in the stimulation of immune cells. A synergistic immunostimulatory effect was observed for BPI in combination with bLP in murine dendritic cells as indicated by the increase of IL-2, IL-12p40 and IL-6 expression (Luminex, n=9). A. Synergism of BPI and bLP in the stimulation of IL-2 expression in murine dendritic cells (upper and lower panel). B. Synergism of BPI and bLP in the stimulation of IL-12p40/p70 expression in murine dendritic cells (upper and lower panel). C. Synergism of BPI and bLP in the stimulation of IL-6 expression in murine dendritic cells (upper and lower panel).

(9) FIG. 7 depicts the binding of BPI to TLR ligands. Binding as well as competitive binding was present for the TLR9 agonist CpG ODN of different types and sequences and for the TLR3 agonist Poly(I:C). A. Streptavidin-coated plates were incubated with biotinylated LPS or biotinylated ODN 1826 (Type B CpG ODN, n=4, meanstandard deviation). Binding of BPI to both LPS and CpG ODN was observed. B. Streptavidin-coated plates were incubated with biotinylated ODN 1826 (n=3, meanstandard deviation). Competition of binding was assessed for different variants of CpG ODNs (ODN 2006 and ODN 2216: Type A CpG ODN, ODN 1585: Type B CpG ODN) and the TLR3 agonist Poly(I:C) in ODN 1826 coated plates.

(10) FIG. 8 depicts binding of lipoarabinomannans and the lipoarabinomanan derivate PiLM by BPI. A. Binding of ManLAM to BPI is shown in microscale thermophoresis (MST) is shown (mean of two replicates). B. Binding of phosphatidyl-inositol lipomannan (PiLM) to BPI is shown in microscale thermophoresis (MST) is shown (mean of two replicates). C. Streptavidin-coated plates were incubated with biotinylated LPS (n=3). Competition of binding was assessed for lipoarabinomannan as exemplified for phosphatidyl-inositol lipoarabinomannan (PiLAM).

(11) FIG. 9 shows that the combination of BPI and CpG ODN induces a synergistic induction of IL-6 and IL12-p70. Bone-marrow derived dendritic cells (BMDCs) were incubated with a combination of BPI (100 nM) and low-dose CpG ODN (ODN 1826, TLR9-agonist, concentration as indicated). A. IL-6 was measured by Luminex 18 h post stimulation (n=4, meanstandard error of the mean). B. IL-12p70 was measured by Luminex 18 h post stimulation (n=4, meanstandard error of the mean). C. Synergistic induction (SI) was calculated for both cytokines (C). For the calculation of SI, the cytokine concentration after adding the combination of both substances was divided by the the sum of cytokine concentrations after stimulation with the single substances.

(12) FIG. 10 shows that the combination of BPI and R848 induces a synergistic induction of IL-6 and IL12-p70. Bone-marrow derived dendritic cells (BMDCs) were incubated with a combination of BPI (100 nM) and low-dose R848 (Resiquimod, TLR7 and TLR8 agonist, nM). A. IL-6 was measured after 18 h (n=3, meanstandard error of the mean). B. IL-12p70 was measured after 18 h (n=3, meanstandard error of the mean). C. Synergistic induction (SI) was calculated for both cytokines (C). For the calculation of SI, the cytokine concentration after adding the combination of both substances was divided by the the sum of cytokine concentrations after stimulation with the single substances.

(13) FIG. 11 shows that the combination of BPI and ManLAM induces a synergistic induction of IL-6 and IL12-p70. Bone-marrow derived dendritic cells (BMDCs) were incubated with a combination of BPI (100 nM) and ManLAM (C-type lectin receptor agonist, 5 M). A. IL-6 was measured after 18 h (n=3, meanstandard error of the mean). B. IL-12p70 was measured after 18 h (n=1, meanstandard error of the mean). C. Synergistic induction (SI) was calculated for both cytokines (C). For the calculation of SI, the cytokine concentration after adding the combination of both substances was divided by the the sum of cytokine concentrations after stimulation with the single substances.

(14) FIG. 12 shows effects of BPI on co-culture of lymphnode cells (LNCs) and dendritic cells (DCs, n=5). BPI stimulates secretion of IFN (Th1), IL-17 (Th17), and does not stimulate secretion of IL-4 (Th2) in this co-culture of LNCs and DCs experiment. Thus, in this setting BPI promotes Th1 and Th17 cell differentiation, and does not promote Th2 cell differentiation. IFN and IL-17 secretion is not observed in a BMDC mono-culture or without addition of BPI to the co-culture of LNC and BMDCs (Medium).

(15) FIG. 13 shows that BPI stimulation activates nave CD4+ and CD8+ T cells. Naive CD4+CD25-CD44-CD62L+ T cells were isolated from peripheral lymph nodes and spleen of C57BL/6J mice and purified by MACS and FACS. Nave CD4+ T cells were cultured in the presence of aCD3 and aCD28 antibodies and dendritic cell-derived supernatant after stimulation with BPI (SN BPI, BPI 200 nM) or after stimulation with ODN 1826 (SN CpG, ODN 1826 20 nM) or after stimulation with BPI and ODN 1826 (SN BPI+CpG, BPI 200 nM, ODN 1826 20 nM) or supernatant without addition of BPI or ODN 1826 (SN NT). On day five, cytokine levels were measured by Luminex. A. Th1 differentiation as shown by detection of IFN (n=3). B. Th17 differentiation as shown by detection of IL-17 (n=3). C. Th22 differentiation as shown by detection of IL-22 (n=3). D. Activation of CD8+ T cells as shown by production of IFN (n=4). Lymph node cells were stimulated with aCD3 and aCD28 antibodies and SN BPI or SN NT and incubated for 6h before adding a protein transport inhibitor cocktail and incubating for additional 16h. Cells were fixed and permeabilized and stained for CD3, CD4, CD8, and IFN. Lines are connecting the amount of IFN-producing CD3+CD8+ T cells after treatment with SN BPI or SN NT for 24h. Bars represent the meansstandard error of the mean. Statistics for comparison were performed with the paired Student's t-test (A to C) or the ratio paired Student's t-test (D).

(16) FIG. 14 shows that SN BPI induces differentiation and proliferation in nave CD4+ T cells (magnification 100). Representative images of nave CD4+ T cell proliferation in T cell stimulation assay shown in FIG. 13 after stimulation with SN BPI or SN NT for one, three or five days.

(17) FIG. 15 is a schematic representation of the T cell activation assay.

(18) In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1

(19) BPI Upregulates Costimulatory Receptors/Maturation Markers in BMDCs

(20) Generation of Recombinant Human BPI

(21) A pCR3 vector (Invivogen) construct comprising an N-terminal HA signal peptide, aa32-487 of the huBPI respectively aa28-483 of muBPI, was transfected into HEK293T cells and used for the experiments after purification.

(22) Generation of GM-CSF Derived Murine BMDCs

(23) BMDCs were generated from the bone marrow of male C57BL/6J mice at the age of approximately 3 to 7 months, which were bred under SPF conditions. BMDCs were generated as previously described [7]. BMDCs were harvested on day 7 by rinsing the dish in order to obtain the non-adherent and loosely-adherent cell fractions.

(24) Cell Stimulation 510.sup.4 BMDCs per well were seeded into a tissue-culture-treated 96-well plate in VLE-RPMI 1640 media (10% FCS, 10% Penicillin-Streptomycin, 50 UM -Mercaptoethanol) and stimulated with 200 nM huBPI.

(25) Fluorescence-Activated Cell Sorting (FACS)

(26) BMDCs were stimulated with huBPI (200 nM) for 18 h. After the stimulation period, the stimulated and unstimulated cells were incubated with Fc-Block and stained using the indicated antibodies (Table 1) and the respective isotype controls (Table 2). 7-Aminoactinomycin D (7-AAD, #00-6993-50, eBioscience) positive stained cells were excluded from the analysis. Flow cytometry measurement was performed using the instrument BD FACS Canto II (BD Bioscience). Data was analyzed using BD FACSDiva software v.7.0 (BD Bioscience) as well as FlowJo v.10 (Tree Star).

(27) TABLE-US-00001 TABLE 1 FACS antibodies Antigen Conjugate Clone Subtype Concentration Catalog-# Manufacturer CD16/CD32 2.4G2 Rat IgG.sub.2b, K 1 ng/L 553142 BD.sup.1 CD11C VioBlue REA754 hu IgG1 3 ng/L 130-110-843 Miltenyi.sup.2 CD80 PE 16-10A1 hamster IgG2K 1.5 ng/L 130-102-883 Miltenyi.sup.2 CD86 FITC PO3.3 rat IgG2bK 1.5 ng/L 130-102-506 Miltenyi.sup.2 CD40 APC FGK45.5 rat IgG2a 3 ng/L 130-102-547 Miltenyi.sup.2 .sup.1BD Biosciences; .sup.2Miltenyi Biotec

(28) TABLE-US-00002 TABLE 2 Isotype controls Antigen Conjugate Clone Subtype Catalog-# Manufacturer Iso REA VioBlue REA293 hu IgG1 parts 130-104-625 Miltenyi.sup.2 Iso hamster IgG2K PE B81-3 Hamster IgG2K 550085 BD.sup.1 Iso rat IgG2b FITC ES265E12.4 Rat IgG2bK 130-103-088 Miltenyi.sup.2 Iso rat IgG2a APC ES26-15B7.3 Rat IgG2aK 130-103-092 Miltenyi.sup.2 .sup.1BD Biosciences; .sup.2Miltenyi Biotec
According to the data presented in FIG. 1, BPI promotes expression of costimulatory receptors CD40, CD80, and CD86, which promote T cell activation.

Example 2

(29) BPI Modulates Cytokine Gene Expression in BMDCs

(30) All methods mentioned in this example were carried out as described in Example 1. BMDCs were seeded into a tissue-culture-treated 96-well plate in VLE-RPMI 1640 media (10% FCS, 10% Penicillin-Streptomycin, 50 M -Mercaptoethanol) and stimulated with 200 nM huBPI or muBPI. 4 h after stimulation, gene expression analysis was performed by microarray analysis (Affymetrix Mouse Gene 2.0 ST, FIGS. 2A and 2B).

(31) Gene Expression Analyses (Quantitative Real-Time PCR)

(32) Gene expression analyses using rtPCR were performed 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h after the stimulation.

(33) After stimulation, cells were lysed and RNA was isolated using RNeasyR Mini Kit (#74106, Qiagen). Remaining DNA in the sample was digested using TURBO DNA-free Kit (#AM1907, ThermoFisher Scientific). Finally, the RNA concentration was measured with a NanoDrop 1000 (Thermo Scientific). The isolated RNA was transcribed into complementary DNA (cDNA) using the iScript Advanced cDNA Synthesis Kit (#1725038, Bio-Rad).

(34) To analyze altered gene transcription, in particular of genes CXCL10, HPRT, IL-2, IL-6, and IL-12p40, in response to different stimulatory reagents, quantitative real-time PCR (qRT-PCR) was performed using SYBR-Green Master Mix (LightCycler 480 SYBR Green I Master, #4707516001, Roche). Gene-specific primers synthesized by Thermo Fisher Scientific, Microsynth, Biomers, and Metabion were used. PCR reaction was performed using the HT 7900 Real-Time PCR system (Applied Biosystems). SDS software version 2.4 (Applied Biosystems) was used to analyze the gene expression, which was normalized to the housekeeping gene HPRT. 2.sup.-CT method was used to analyze the data.

(35) Both human and murine BPI shows an immunostimulatory effect on BMDCs (FIG. 2A). Upon stimulation with BPI BMDCs an increase of CXCL10, IL-2, IL-6, and IL-12p40 mRNA expression is seen (FIGS. 2B and C).

Example 3

(36) BPI Modulates Cytokine Protein Secretion by BMDCs

(37) All methods mentioned in this example were carried out as described in the preceding examples. Cells were stimulated with Zymosan depleted (Zymd, 5 g/ml) from Invivogen (San Diego, CA, USA) or (R)-Pam.sub.3CSK.sub.4 (bLP, 10 nM) from EMC Microcollections GmbH (Tbingen, Germany). Protein secretion analysis was performed using the supernatants.

(38) Protein Quantification by Luminex Technology

(39) To quantify the interleukin and chemokine protein-level after stimulation with huBPI, Zymd, and (R)-Pam CSK.sub.4, a multiplex-system was used and determined by using the Luminex technology (Austin, TX, USA). The cytokines were captured with anti-mouse capture-antibodies and detected by biotinylated antibodies specific for the respective protein (Table 3). Protein concentrations were calculated using a reconstituted standard curve of the analyzed proteins (SM039, #LMC4031, Lot #1438354; Thermo Fisher Scientific; Standard Mix 1a, #EPX010-20603-901, Lot #111709101, eBioscience).

(40) TABLE-US-00003 TABLE 3 Material for protein quantification of the different proteins by ELISA or Luminex technology Cytokine Antibodies Manufacturer CXCL10 ELISA Set, #900-M153 Peprotech IL-2 #554424, #554426 BD Biosciences IL-6 ELISA Set, #555240 BD Biosciences IL-12p40/p70 #551219, #554476 BD Biosciences TNF ELISA Set, #558534 BD Biosciences
BPI shows an immunostimulatory effect on BMDCs, such as increase of IL-2, IL-12p40/p70, and IL-6 protein secretion (FIG. 3). The increase of IL-2 and IL12p40/p70 is significantly higher compared to Zymd and bLP, whereas the IL-6 level is comparable. Therefore, BPI is a very effective inductor of IL-2, which is a central cytokine for induction of lymphoid cells such as Th cells and CD8+ T cells.

(41) Protein levels of proteins involved in T cell activation (IL-2), and of proteins involved in promotion of Th1 cells (IL-2, IL-12p40/p70), Th17 cells (IL-6), Th22 cells (IL-6 and TNF), and cytotoxic T cells (IL-2), were increased after BPI treatment (FIG. 3 and FIG. 4), whereas the IL-4 level was not induced by BPI in BMDCs (FIG. 12).

Example 4

(42) BPI Preferentially Stimulates Subtypes of Antigen-Presenting Cells as Shown Comparing Murine Dendritic Cells and Macrophages

(43) All methods mentioned in this example were carried out as described in Example 1 and 2.

(44) Generation of BMDM was carried out similarly to BMDCs; MCSF was used instead of GMSCF for differentiation of BMDM. Cells were re-plated on day 1 of culture and harvested on day 5. On day 1, cells were seeded in new plates and harvested on day 5 for the experiments. Cells were stimulated with LPS Ec (10 ng/ml) or BPI (100 nM) for 18 h before supernatants were collected. TNF levels were determined by ELISA (OPTEIA Murine TNF ELISA Set, BD Bioscience, Heidelberg, Germany).

(45) Dendritic cells secrete TNF in response to stimulation with BPI and LPS Ec, whereas macrophages secrete TNF in response to stimulation with LPS Ec but not in response to stimulation with BPI (FIG. 4).

(46) Accordingly, BPI preferentially stimulates antigen-presenting cells such as dendritic cells ensuring an efficient antigen-presentation and a reduction of side effects caused by stimulation of other cells when used in vivo.

Example 5

(47) BPI Binds to bLPs and LTA, and Shows a Synergistic Immunostimulatory Effect in PBMCs when Coadministered

(48) MicroScale Thermophoresis Experiments

(49) BPIN (A) and rBPI were labeled with NT647 in PBS pH 7.4 (Monolith NT Protein Labeling Kit RED-NHS, NanoTemper Technologies, Munich, Germany). Concentration of labeled protein was determined using the NanoDrop (ThermoScientific, Wilmington, NC, USA) and Bradford assay (Promega, Mannheim, Germany). MST binding experiments were carried out with 5 nM labeled protein in binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.1% Tween) with 0.196-6,436 nM of Pam.sub.3CSK.sub.4 (bLP), or 0.61-20,000 nM of LTA SPlgt (LTA), or >0.61-20,000 nM WTA SPlgt (WTA) at 20%-40% MST power, 20% LED power in premium capillaries on a Monolith NT.115 pico device at 25 C. (NanoTemper Technologies, Munich, Germany). Pam.sub.3CSK.sub.4, (bLP) was obtained from EMC Microcollections GmbH (Tbingen, Germany), LTA and WTA were prepared as published previously [8]. MST timetraces were recorded, and the temperature jump and thermophoresis or thermophoresis was analyzed. Binding graphs of each independent experiment were normalized to the fraction bound.

(50) Isolation and Stimulation of Human Peripheral Blood Mononuclear Cells

(51) After informed consent, blood was drawn from healthy male volunteers using heparinized blood collection tubes and PBMCs were separated using leucosep tubes containing FICOLL PAQUE PLUS (Oxford Immunotec, Abingdon, Great Britian) at 1,000g for 10 min. The pellet was resuspended in AIM VR Medium (Thermo Fisher Scientific Inc., Waltham, MA, USA), counted, and cultivated in 96-well plates for 4 h (110.sup.5/100 l). Then cells were stimulated with (R)-Pam.sub.3CSK.sub.4 (bLP, 1 nM) or LTA S. pneumoniae lgt (a lipoprotein deficient strain, LTA SPlgt, 1 M, n=4) in the presence or absence of BPI (500 nM). The supernatants were collected after 18 h for determination of cytokine concentration by ELISA according to the manufacturer's instructions (OPTEIA Human TNF ELISA Set, BD Biosciences, Heidelberg, Germany).

(52) BPI was shown to bind to bLPs. Affinity of BPI to bLP was within a nanomolar range (FIG. 5A). BPI was also shown to bind to lipoteichoic acids (LTAs). LTAs are another major component of the cell wall of Gram-positive bacteria and is anchored to the cell membrane via a lipophilic anchor. As representative samples, LTA preparations of S. pneumoniae lgt (a lipoprotein deficient strain, LTA SP lgt) were tested for their BPI binding potential. Despite a conserved structure compared to LTAs, WTAs lack the acyl-anchor of LTAs. MST did not show binding of WTA preparations of S. pneumoniae lgt (WTA SP lgt) demonstrating specificity of the BPI binding to LTA and its acyl-anchor (FIG. 5). Thus, BPI binds ligands such as LTA in dependence of an acyl-anchor.

(53) Coadministration of BPI and a bLP as well as LTA SP showed a synergistic immunostimulatory effect in upregulation of several cytokines, such as TNF (FIGS. 5B and D).

(54) Accordingly, the present inventors disclose TLR ligands originating from Gram-positive bacteria as new ligands of BPI. Furthermore, BPI as well as the TLR ligands bLPs and LTAs have a synergistic immunostimulatory effect on PBMCs. Thus, BPI does not only bind to TLR ligands, but also has a synergistic effect when coadministered with a TLR ligand even when BPI alone has a minor effect on this cell type. This indicates interaction and synergistic effects of BPI with ligands displaying an acyl-anchor.

Example 6

(55) BPI and TLR Ligands Show a Synergistic Immunostimulatory Effect in BMDCs

(56) All methods mentioned in this example were carried out as described in the preceding examples. (R)-Pam.sub.3CSK.sub.4 (bLP, 10 nM) was obtained from EMC Microcollections GmbH (Tbingen, Germany). The TLR9 ligands CpG ODNs (ODN 1826, ODN 2006, ODN 1585, ODN 2216), were from Biomers (Ulm, Germany) and the TLR7/8 ligand R848 was purchased from Invivogen (San Diego, CA, USA).

(57) Coadministration of BPI and a bLP showed a synergistic immunostimulatory effect in upregulation of several cytokines, such as IL-2, IL-12p40/p70, and IL6 (FIG. 6). Coadministration of BPI and the TLR9 ligand CpG ODN (ODN 1826) showed a remarkably high synergistic immunostimulatory effect in upregulation of several cytokines, such as IL-12p70 and IL6 (FIG. 9). Coadministration of BPI and the TLR7/8 ligand R848 also showed a synergistic immunostimulatory effect in upregulation of several cytokines, such as IL-12p70 and IL6 (FIG. 10).

(58) Accordingly, BPI in combination with TLR ligands as exemplified for the TLR2 ligand bLP, the TLR9 ligand CpG ODN and the TLR7/8 ligand R848 synergistically augments expression and secretion of proteins stimulating Th1 cells, Th17 cells, and cytotoxic T cells compared to stimulation with BPI or TLR ligand only.

Example 7

(59) BPI and CLR Ligands Show a Synergistic Immunostimulatory Effect in BMDCs

(60) All methods mentioned in this example were carried out as described in the preceding examples. Mycobacterial ManLAM was obtained from Nacalai Tesque (Kyoto, Japan).

(61) Coadministration of BPI and the CLR ligand ManLAM showed a synergistic immunostimulatory effect in upregulation of several cytokines, such as IL-12p70 and IL6 (FIG. 11).

(62) Accordingly, BPI in combination with the CLR ligand ManLAM synergistically augmented expression and secretion of proteins stimulating Th1 cells, Th17 cells, and cytotoxic T cells compared to stimulation with BPI or ManLAM only.

Example 8

(63) BPI Binds to TLR and CLR Ligands

(64) MST was performed as described for example 5. PiLAM and PiLM were ordered from Invivogen (San Diego, CA, USA). BPI binding assays were performed by incubating Streptavidin-coated 96-well plates (Nunc Immobilizer Streptavidin F96 clear, Thermo Fisher Scientific, Waltham, MA, USA) with LPS biotin (2 g/ml) in PBS overnight at 23 C. with gentle agitation. After washing with assay buffer (150 mM NaCl, 50 mM HEPES, Sigma Aldrich, Taufkirchen, Germany) containing 0.01% Casein, plates were blocked with 10 g/l BSA (Sigma Aldrich, Taufkirchen, Germany) at 37 C. and washed again. Thereafter, ligands were preincubated with 20 nM BPI in assay buffer containing 1 g/l BSA for 30 min and loaded onto the plates. Bound BPI was detected by murine anti-human BPI monoclonal antibody (Cat.-No. HM2042, Hycult Biotech, Uden, Netherlands) and HRP-conjugated rabbit anti-mouse IgG (Cat.-No. 315-035-048, Dianova, Hamburg, Germany). TMB (BD OptEIA TMB Substrate Reagent Set, BD Biosciences, Heidelberg, Germany) was used as a substrate for peroxidase. After addition of 1 N HCl, absorbance was measured at 450 nm. Absorbance indicates binding, the higher the absorbance the higher the binding of BPI to the coated well. Preincubation with a BPI ligand inhibits binding in a concentration-dependent manner.

(65) BPI was shown to bind to bLPs and lipoteichoic acids (LTAs) in FIG. 5. Additionally, the TLR9 ligands CpG ODNs, the TLR3 ligand Poly(I:C) and the CLR-ligand ManLAM bind to BPI (FIGS. 7 and 8). Additionally, as seen for bacterial lipopeptides and lipoteichoic acids (FIG. 5), binding is conserved for lipomannans and other lipoarabinomannans such as PiLAM containing an acyl-anchor (FIG. 8).

(66) Accordingly, BPI binds to ligands containing an acyl-anchor as exemplified for TLR and CLR ligands such as bLPs, LTAs, ManLAM, PiLAM and PiLM. BPI also binds to ligands consisting of nucleic acids and analoga thereof as exemplified for TLR ligands such as CpG ODNs or Poly(I:C).

Example 9

(67) BPI-Stimulated Dendritic Cells Induce IFN and IL-17 Production in Peripheral Lymph Node Cells

(68) All methods mentioned in this example were carried out as described in the preceding examples. Peripheral lymph nodes of C57BL/6J mice were collected to obtain lymph node cells. The cells were isolated by pressing the lymph nodes through a 22 m cell strainer, flushing the homogenate with either RPMI-VLE or PBS, and collecting the cell dispersion in a 50 ml falcon tube. The suspension was centrifuged (300g, 21 C., 8 min) and the cell pellet was resuspended in 2 to 5 ml of cell culture medium (VLE-RPMI 1640 medium containing glutamine supplemented with 10% heat-inactivated FCS, 10% penicillin-streptomycin and 50 M sterile-filtered -Mercaptoethanol). After this procedure the cell suspension was washed once or twice in cell culture medium or PBS by centrifugation (300g, 21 C., 8 min). BMDCs were seeded and co-cultured with a threefold excess of peripheral lymph node cells in the presence or absence of BPI (200 nM) in in cell culture medium. Supernatans were collected and measured as described in preceding examples.

(69) Significant secretion of IFN and Il-17 was only seen in the presence of BPI (FIG. 12). Secretion of IL-4 was not observed in any of these settings.

(70) This experiment indicates that BPI indeed promotes the activation of Th1 and Th17 cells and/or other IFN and IL-17 producing cells in the co-culture of dendritic cells and lymph node cells.

Example 10

(71) Supernatant of BPI-Stimulated Dendritic Cells Induce IFN and IL-17 Production in CD4+ and CD8+ T Cells

(72) Isolation and Sorting of CD4+ Cells

(73) To isolate CD4+SC and pLNC, cells isolated from spleen and peripheral lymph nodes (see 2.1.3) were sorted by magnetic cell sorting (MACS), using a CD4+ T cell isolation kit (#130-104-454, Miltenyi Biotec). Isolation was performed according to the manufacturer's protocol. In detail, cells were labeled with biotin-antibodies against CD8 and several non-lymphocyte markers and magnetic anti-biotin beads. CD4+ cells were then isolated by applying the cell suspension onto a LS column inside a strong magnetic field of a MACS separator. The flow-through, containing unlabeled CD4+ cells, was collected and cell number was determined using a hemocytometer. CD4+SC and pLNC were used in experiments or labeled with fluorescent antibodies against non-nave T cell markers for fluorescence activated cell sorting (FACS) of nave CD4+ T cells. To isolate nave CD4+ T cells by FACS, CD4+SC/pLNC where resuspended in 100 l of cold FACS buffer (PBS, 1% FCS, 0.05% sodium azide (#1.06688, Merck)) per 210.sup.6 cells. Cells where then stained with fluorochromes against T cell markers (25 min, 4 C., protected from light). After staining, cells where washed two times with 2 ml of PBS (800 g, 4 C., 8 min) and then resuspended in 1 ml of cold PBS. Cells where then applied to a 30 M pre-separation filter (#130-041-407, Miltenyi Biotec) and sorted using FACS Aria. Nave CD4+ T cells were identified by a CD4-positive, CD25-negative, CD44-negative and CD62L-positive (CD4+CD25-CD44-CD62L+) phenotype. Fluorescent antibodies used are listed in Table4.

(74) T Cell Activation Assays

(75) T cell activation assays were performed in a 96-well, 48-well or 12-well clear flat bottom TC-treated culture microplate (#353072, #353230, #353225, Falcon), which where coated at 37 C. for 1-2h with a 1:400 dilution of purified hamster anti-mouse aCD3 (#553057, BD) in sterile PBS. After coating, wells where washed twice with the same volume of PBS used for coating. Then, purified hamster anti-mouse aCD28 (#553294, BD) antibodies were diluted 1:250 in respective cell suspensions, e.g. nave CD4+ T cells suspended in VLE-RPMI 1640 medium containing glutamine supplemented with 10% heat-inactivated FCS, 10% penicillin-streptomycin and 50 M sterile-filtered -Mercaptoethanol, and suspensions were applied into coated wells. Supernatans were collected and measured as described in preceding examples.

(76) Intracellular Staining of Surface Markers and Cytokines

(77) All described work steps were conducted on ice. To stain cytokines and surface markers intracellularly, isolated SC, pLNC or mLNC were permeabilized and fixed after stimulation in a T cell activation assays. To avoid secretion of cytokines during incubation, cells were additionally treated with a commercial protein transport inhibitor cocktail (#00-4980-03, ThermoFischer). After seeding, cells were re-stimulated with SN BPI or SN NT and incubated for 6h. For further incubation, protein transport inhibitor cocktail was added to a final concentration of 0,5 and cells were incubated for additional 18 h. After incubation, cells were rinsed from the well and collected in a round bottom polystyrene test tube (#352058, Falcon). Cells were then fixed and permeabilized using the BD Transcription Factor Buffer Set (#562574, BD). In more detail, cells were washed with 1 ml of cold PBS (800 g, 4 C., 8 min) and resuspended in 1 ml of cold PBS. The cell suspension was then applied onto a 30 M pre-separation filter (#130-041-407, Miltenyi Biotec) and collected in the same tube. Cells were then stained with a fixable viability stain (#565388, BD) for 30 min. After staining, cells where washed twice with 2 ml of FACS buffer (300 g, 4 C., 8 min) and then resuspended in 100 l of FACS buffer. To avoid background staining, cells were treated with FC block in a 1:500 dilution for 10 min in the dark and were then washed two times 1 ml FACS buffer (300 g, 4 C., 8 min). Cells were then fixed and permeabilized for 45 mins in 1 ml fixation/permeabilization reagent. After fixation, cells were washed twice with 1 ml of perm/wash solution (350 g, 4 C., 5 min) and stained with fluorescent antibodies and respective isotype controls (Table4).

(78) TABLE-US-00004 TABLE 4 Fluorescent antibodies used for FACS analysis and sorting Antigen Catalog# Manufacturer CD3 663066 BD Biosciences CD4 553052 BD Biosciences CD8 1550-02 Southern Biotec CD25 130-120-697 Miltenyi Biotec CD44 553133 BD Biosciences CD62L RM4304-3 Caltag INF 554412 BD Biosciences
Protein Quantification by Luminex Technology

(79) Measurements of cytokines was performed as described in example 3 except for using antibodies specific for IFN, IL-17 and IL-22 as depicted in Table 3.

(80) TABLE-US-00005 TABLE 5 Material for protein quantification of the different proteins by ELISA or Luminex technology Cytokine Antibodies Manufacturer IFN #MAB785, #554410 R&D systems, BD Biosciences IL-17A #555068, #555067 BD Biosciences IL-22 ELISA Set, #88-7422-88 Invitrogen

(81) This experiment indicates that BPI alone or in combination with CpG ODNs indeed promotes not only activation but also differentiation of nave T cells into Th1, Th17 and Th22 cells as well as activation of CD8+ T cells. Th1 differentiation is especially promoted by stimulation with BPI in combination with CpG ODNs.

Example 11

(82) As shown in FIG. 15, antigen-presenting cells, preferably BPI high-responsive cells, e.g. DCs, are stimulated with BPI alone or in combination with BPI ligands or other suitable combination partners, e.g. bLPs, LTAs, CpG ODNs, R848, Poly(I:C) or ManLAM. After variable incubation time, e.g. for 1 hour to 4 weeks, supernatants (SN) are collected, pooled and stored at 20 C. T cells are isolated from peripheral blood or other organs of a donor. For T cell activation, T cells are stimulated with the supernatant (SN) in combination with other stimuli e.g. aCD3 antibodies, aCD28 antibodies, specific antigens, dendritic cells, other antigen-presenting cells or a combination thereof. After a variable incubation time, e.g. from 1 hour to 3 month, differentiated T cells are harvested for transfer to patients or storage. As examples for suitable methods see examples 1 to 7 (FIGS. 1 to 5 and 6 to 11, stimulation of antigen-presenting cells), example 9 (FIG. 12, stimulation of lymph node cells in the presence of dendritic cells) and example 10 (FIG. 13, stimulation of CD4+nave T cells and CD8+ T cells with supernatant derived from dendritic cells stimulated with BPI or BPI in combination with BPI ligands).

(83) The present inventors have surprisingly found that BPI can activate immune cells, such as dendritic cells, even without interaction partners, particularly without microbial ligands, resulting in a BPI-specific pattern of gene expression and protein secretion. Administration of BPI to target cells unexpectedly resulted in expression of T cell activating and Th1 cell, Th17 cell, and cytotoxic T cell promoting cytokine IL-2, and other immunostimulatory cytokines. IL-2 expression was extraordinary high as compared to TLR and CLR ligands. Furthermore, BPI preferentially stimulates antigen-presenting cells such as dendritic cells. Moreover, the present inventors disclose the immunostimulatory effect of BPI to be synergistically enhanced by TLR- and CLR-ligands, such as bacterial lipopeptides, lipoteichoic acid, nucleic acids and nucleic acid analoga. The molecules regulated by BPI, such as cytokines, chemokines, cell surface molecules, and receptors, have a high potential to regulate immune cells, and thus the immune response to endogenous and exogenous antigens. Conclusively, BPI has a high potential to be used in a method of immunization, or in combination with other suitable combination partners and/or an antigen. BPI preferentially stimulates antigen-presenting cells such as dendritic cells unexpectedly resulting in high secretion of IL-2 and CXCL10, which consecutively induce immune cells, such as Th1 cells and cytotoxic T cells. Therefore, BPI is a very promising candidate for an adjuvant used in a vaccination against HIV, hepatitis, influenza, malaria, Mycobacterium tuberculosis, allergies, or cancer.

(84) The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.

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