NEW ADJUVANT TO IMPROVE THE INNATE IMMUNITY

Abstract

The present invention relates to the field of adjuvant and vaccination. In the present study, the inventors investigate whether P1, in addition to being an antigen, could act as an adjuvant by first exploring its capacity to stimulate epithelial TSLP production. They evaluated additional immunomodulatory effects of P1 on human nasal mucosal models, including cytokines and chemokines production, intracellular signaling pathways, mucosal DC activation, T cell proliferation, and antigen-specific B cell responses against a model antigen in vitro. Altogether, they reported the immunological mechanism underlying P1-vaccine and the interest of P1 as a nasal mucosal adjuvant. Thus, the present invention relates to an immunoadjuvant composition comprising the P1 peptide of the HIV-1 envelope subunit gp41.

Claims

1. An immunoadjuvant composition comprising the P1 peptide of the HIV-1 envelope subunit gp41.

2. A method of improving the innate immunity of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immunoadjuvant composition of claim 1, or a nucleic acid encoding the P1 peptide of the HIV-1 envelope subunit gp41.

3. (canceled)

4. A method of treating an infectious disease in a subject in need thereof comprising administering to the subject i) P1 peptide of the HIV-1 envelope subunit gp41 as an adjuvant, and ii) a treatment for the infectious disease.

5. The method of claim 4, wherein the P1 peptide of the HIV-1 envelope subunit gp41 and the treatment against the infectious diseases are administered as a combined preparation for simultaneous, separate or sequential use.

6. The according to claim 4, wherein the infectious disease is caused by a pathogen which is a virus, a bacteria or a parasite.

7. The immunoadjuvant composition according to claim 1 wherein the peptide P1 has a sequence set forth as in SEQ ID NO: 1.

8. The immunoadjuvant composition according to claim 1, wherein the peptide P1 has a sequence set forth as in SEQ ID NO: 2, 3 or 4.

9. (canceled)

10. A vaccine composition, comprising at least one antigen and at least the P1 peptide of the HIV-1 envelope subunit gp41 and, optionally, one or more pharmaceutically acceptable excipients.

11. A method of improving the innate immunity of a subject in need thereof comprising administering to the subject a therapeutically effective amount of the vaccine composition of claim 10.

12. A method of treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the vaccine composition of claim 10.

13. (canceled)

Description

FIGURES

[0091] FIG. 1. P1 induces TSLP expression in nasal epithelial cells. (A) Confluent RPMI 2650 cells were cultured with P1 peptide (5 M, 25 M, 125 M) or scramble peptide (125 M), for 2 hours or 4 hours. TSLP secretion in culture supernatants was quantified by ELISA. (B) RPMI nasal cells were cultured with HIV-1 Glade A, B or C derived P1 peptides or the mutated P1-5W peptide at increasing concentrations for 4 hours. Inset: P1 key amino acids (661-670) corresponding to the broadly neutralizing 2F5 and 4E10 IgG epitopes with Glade-specific mutations are aligned. (C) RPMI nasal cells were pre-incubated with anti-galactosyl ceramide (GalCer) antibody for 30 min at 37 C. before stimulation with each P1 peptide. (D) Primary HNEC cells were cultured with P1 peptide (5 M, 25 M, 125 M), with or without anti-GalCer pre-incubation, or P1W peptide (125 M). Data are presented as meanSEM (n=3-8 independent experiments; paired student's t-test *p<0.05, **p<0.01 ***p<0.001, ****p<0.001).

[0092] FIG. 2. In vitro immunization with ovalbumin adjuvanted by P1 peptide. (A): CD8-depleted PBMCs were cocultured with RPMI nasal cell monolayer for 24 hrs prior to addition of OVA as an antigen, adjuvanted by P1 at three concentrations or in the presence of mutated P1 (P1mut), in the absence of antigen or adjuvant as negative controls. CD20+ B cells expressing OVA-specific IgA or IgG were quantified by flow cytometry using anti-CD2O-PE, FITC-conjugated OVA, and APC conjugated anti-human-IgA or anti-human-IgG. Data are presented as meanSEM (n=5 independent donors, paired student's t-test *p<0.05, p<0.01). B and C: Mice were immunized with P1 or mutated P1 by the intra-nasal (IN) route. Resulting mucosal IgA response was evaluated in the feces after three immunizations by ELISA; unpaired student's t-test: *p<0.05 (B). Resulting OVA-specific CD8+ T cell response was evaluated by flow cytometry after staining with OVA-peptide SIINFEKL pentamers; unpaired student's t-test: **p<0.01 (C).

EXAMPLE

[0093] Material & Methods

[0094] Peptides

[0095] Peptide P1 (aa 650-685) is derived from HIV-1 gp41 envelope subunit. P1 Glade B (SQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK, SEQ ID NO: 3) was derived from the Clade B HXB2 isolate; P1 Glade A (SQIQQKKNEQDLLALD KWANLWNWFDISNWLWYIR, SEQ ID NO: 2) from the Glade A 99UGA07072 isolate, and P1-clade C (SQTQQEKNEQELLALDSWKNLWNWFSITNWLWYIK, SEQ ID NO: 4) was derived from the Clade C Bw96Bw0502 isolate. P1W is a P1 Glade B variant with W666G mutation and P1-5W with all five W mutated to G. Scramble peptide sequence comprised the same set of amino acids found in P1 Glade B but organized in a random manner (Alfsen and Bomsel 2002). Peptides were synthesized with a purity >95% by Biopeptide Co., Inc (San Diego, CA) or United BioSystems (VA, USA).

[0096] Cells

[0097] Nasal RPMI 2650 cells (isolated from the human nasal septum, squamous cell carcinoma, ATCC) were grown in MEM (Minimum Essential Medium , Thermo Fisher) supplemented with 10% fetal calf serum (FCS, Eurobio, Courtaboeuf, France) and 1% penicillin/streptomycin.

[0098] Primary human nasal epithelial cells (HNECs, purchased from PromoCell, Heidelberg, Germany) were isolated from nasal septum or adenoids of healthy donors. Cells from two independent donors were obtained. HNECs were cultured in airway epithelial cell basal medium (PromoCell) and supplemented with airway epithelial cell growth SupplementMix (PromoCell) and only cells from passage 2 to 6 were used.

[0099] Monocyte-derived DCs (DCs) were generated from primary human monocytes obtained from PBMCs (purity>98%) as described (Sallusto and Lanzavecchia 1994, Magrus-Chatinet, Yu et al. 2007). In brief, human peripheral blood mononuclear cells (PBMC) were separated from healthy donors blood (EFS, Paris, France), and monocytes were purified from PBMC by negative selection according to manufacturer instructions (StemCell Technologies, France). DCs were obtained by incubating monocytes for 7 days in complete medium containing GM-CSF (100 ng/ml) and IL-4 (long/ml).

[0100] Autologous CD4+ T cells were purified from PMBCs by negative selection according to manufacturer instructions (StemCell Technologies, France) (purity>95%)

[0101] Quantitative RT-PCR for TSLP

[0102] The expression of short and long form TSLP was quantified as described (Bjerkan, Schreurs et al. 2015, Dong, Hu et al. 2016). Briefly, total RNA was extracted using Trizol. Five hundred nanograms of RNA were treated with ezDNase Enzyme (Thermo Fisher) to remove genomic DNA, and reverse transcribed into cDNA using the kit SuperScript IV VILO Master Mix according to manufacturer instructions (Thermo Fisher). Quantitative PCR was performed using reported primers (Dong, Hu et al. 2016) and the PowerUp SYBR Green Master Mix according to manufacturer instructions (Thermo Fisher). Reactions were performed in triplicates, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. Amplification, data acquisition, and analysis were carried out using the LightCycler 480 Software (Roche, Mannheim, Germany). The levels of TSLP mRNA were normalized to the levels of GAPDH using the Ct method (Schmittgen and Livak 2008) and were presented as 2-Ct values.

[0103] MicroRNA Microarray Analysis

[0104] Confluent HNECs in 12-well plate were stimulated with medium or P1 (clade B, 125 M) for 6 hours, at 37 C. Total RNA was extracted using Trizol. Before analysis, lfTSLP RNA up-regulation was confirmed by qPCR as described above and RNA quality was assessed with Agilent 2100 bioanalyzer according to manufacturer instructions (Agilent Technologies). Three untreated and treated paired samples from three independent experiments were analyzed by GeneChip miRNA 4.0 arrays (Affymetrix, Thermo Fisher) containing probes for 2578 human mature microRNAs and 2025 pre-mature microRNAs (https://assets.thermofisher.com/TFS-Assets/LSG/brochures/miRNA_4-0_and_4-1_datasheet.pdf). Potential microRNA targets were analyzed with the Ingenuity Pathway Analysis (IPA) software (Qiagen).

[0105] MiR-4485 Quantification and Knockdown

[0106] The quantification and knockdown of microRNA were performed as previously described with some modifications (Zhou, Xu et al. 2018). Briefly, total RNA was purified using MinElute PCR Purification Kit (Qiagen), the expression level of miR-4485 was quantified with TaqMan Small RNA Assays (Thermo Fisher). Reactions were performed in triplicates, and U6 was used as endogenous control. In order to knockdown miR-4485, 70% confluent HNEC cells were transfected with anti-miR-4485 inhibitor (67 nM, Qiagen) or mock inhibitor (miSCRIPT inhibitor negative control, 67 nM, Qiagen) using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer. 36 h after transfection, miR-4485 expression, when quantified as described above, was reduced by 50-60% in anti-miR-4485 transfected cells as compared to anti-miR control (n=3 independent experiments).

[0107] Signaling inhibitors Confluent HNEC cells in 24-well plate were pre-incubated with inhibitors for 1 h at 37 C. prior to P1 treatment. Inhibitors, namely dexamethasone (Dex) a NF-kB and MAPK inhibitor (used at 100 nM), and Cyclosporin A (CsA) a Calcineurin Inhibitor (used at 1.5 M), were from Invivogen and used at the manufacturer's recommended concentrations. ENMD-1068 (PAR-2 antagonist, Enzo Life Science) was used at 500 g/mL as described (Kelso, Lockhart et al. 2006, Wygrecka, Dahal et al. 2013).

[0108] Calcium Measurement

[0109] 70-80% confluent RPMI 2650 or HNEC cells in 24-well plate were loaded with 2 mM Fura-2/AM (Molecular Probes) in basal medium without serum/growth factors for 1 hour at 37 C. Cells were washed twice with mammalian saline (Conche, Boulla et al. 2009) and measurements were performed in complete medium supplied with HEPS (10 mM) and CaCl2 (2 mM) as described add (Conche, Boulla et al. 2009). Images were acquired with an inverted fluorescence microscope (Observer Z1, Zeiss, Germany) and analyzed with MetaMorph software (Guichard, Bonilla et al. 2017) Calcium was measured every 5 seconds by video fluorescence imaging. Results were expressed as 340 nm to 380 nm fluorescence ratio and normalized to the baseline, i.e. ratio at time zero was set as 1.

[0110] Cytokines and Chemokines Quantification

[0111] TSLP, IL-25/IL-17E, IL-33, IFN-, IL-10, IL-12/23p40, IL-4, IL-5, IL-6, IL-13, TNF-, MMP-9, IL-8/CXCL8, MIP-3/CCL2, MCP-1/CCL20, MDC/CCL22, TARC/CCL17, APRIL and BAFF were measured in culture supernatants from indicated experiments with custom multiplex Luminex assays (Bio-techne) according to the manufacturer's instructions. Additionally, when indicated TSLP was measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 8 pg/ml (Thermo Fisher) according to the manufacturer's instructions.

[0112] DC-EC Co-Culture and DC Activation

[0113] Three DC culture systems were developed. Monocytes derived DCs (5105 cells) were incubate for 24 h in medium alone and considered as non-mucosal DCs (DCs) or co-cultured with nasal epithelial cell (RPMI-2650 cell line) monolayer in 24-well plate (DC-EC or eduDC systems for 24 hours at 37 C. In turn, DCs were either further cultured with ECs during P1 stimulation (DC-ECs) or separated from EC and transferred into a new plate (eduDCs) for further P1 treatment. Subsequently, P1 (clade B, 125 M) or medium were added to each of the DCs, DC-ECs or eduDCs cultures for 16 hrs. DCs were collected for surface staining with allophycocyanin (APC)-conjugated anti-CD86, R-phycoerythrin (PE)-conjugated anti-CD83, APC-conjugated anti-TSLPR, PE-conjugated anti-IL-7R antibodies (all from Bio-Techne). Specific labeling was quantified by flow cytometry using a Guava EasyCyte flow cytometer and the InCyte software (Merck) described (Duchemin, Khamassi et al. 2018). Culture supernatant were collected and frozen at 80 C. for subsequent cytokine and chemokine analyses.

[0114] DC-T Co-Cultures

[0115] DCs and confluent ECs were co-cultured overnight as described above, and DC-EC or eduDC was further incubated with P1 (clade B, 125 M) or medium for 24 h. Then, DCs were separated and incubated with autologous CD4+ T cells pre-labeled with CFSE (Thermo Fisher) according to the manufacturer's instructions, at a ratio of 1:5 (DC/T). After 5 days of culture, CD4+ T cell proliferation was analyzed by flow cytometry as described (Yeh, Yeh et al. 2013, Qin, Yin et al. 2015) using Phytohaemagglutinin (PHA) (5 g/mL) as positive control.

[0116] In Vitro Immunization Assay

[0117] In vitro immunization assay was performed as reported (Jung, Matsumoto et al. 2007) with modifications. Briefly, 1106 CD8-depleted PBMCs (Human CD8 Depletion Cocktail, StemCell Technologies, France) were cocultured for 24 hrs with RPMI 2650 cells (1105) pre-seeded in 48 well plates for 48 hrs. Then, ovalbumin (OVA, EndoFit Ovalbumin, 10 g/mL, Invivogen) alone, OVA together with P1 (5 M, 25 M, 125 M), OVA together with P1 mutant (P1mut, 125 M), or medium were added to in RPMI 1640 medium supplemented with Non-Essential Amino Acids (NEAA solution, Thermo Fisher), IL-4 (long/mL), IL-2 (10 UI/mL) and 2-mercaptoethanol (20 M) for 7 days.

[0118] For the detection of OVA-specific B cells, at indicated time of culture, PMBCs were surface stained with ovalbumin conjugated to fluorescein (OVA-FITC, 20 ug/mL, Thermo), PE-conjugated mouse anti-human CD20 (BD Biosciences, CA, USA), APC-conjugated goat anti-human IgA or donkey anti-human IgG (Jackson ImmunoResearch, PA, USA) as indicated by the manufacturer. Specific labeling was quantified by flow cytometry with a Guava EasyCyte flow cytometer (Merck-Millipore), and analyzed with the dedicated InCyte software, using the following strategy: CD20+ B cells were first gated and cell double positive for OVA-FITC+ and APC-conjugated anti-IgA or anti-IgG were determined as OVA-IgA or IgG-specific B-cell s.

[0119] Statistical Analysis.

[0120] Data are presented as meanSEM of at least three independent experiments. Statistical significance was analyzed by the two-tailed Student's t-test with the GraphPad Prism software.

[0121] Results

[0122] P1 Induces TSLP Secretion in Nasal Epithelial Cells by Interacting with Galactosyl Ceramide.

[0123] We first investigate whether P1 induced TSLP secretion in nasal epithelium. Therefore, we cultured human nasal epithelial cells (RPMI 2650) with P1 Glade B for 2-24 hours at 37 C. and analyzed the culture supernatants for TSLP secretion. Compared with the medium and scramble peptides used as negative controls, P1 upregulates TSLP secretion in a dose-dependent manner from 2 h to 4 h (FIG. 1A). At 125 M, when P1 adopts a trimeric oligomerization state (Alfsen and Bomsel 2002), P1 induces a significantly higher secretion of TSLP than in a monomeric state (at 5 M, and 25 M). TSLP secretion occurs rapidly within hours post stimulation reaching a plateau from 4 to 24 h.

[0124] Although P1 sequence is relatively conserved, in contrast to highly mutated regions of HIV-1 envelope gp120, P1 sequence varies between HIV-1 Glade A that is common in West Africa, Glade B that predominates in Europe and the USA, and Glade C that predominates in Africa and China (FIG. 1B). Consequently, we next analyzed whether TSLP secretion was restricted to Glade B derived P1, or would also be stimulated by P1 derived from Glade A and C viruses (FIG. 1B). Secretion of TSLP induced by Clade A compared to Glade B P1 is reduced by 20% (41.82.6 pg/mL for Glade A, 52.33.4 pg/mL for Clade B P1 at 125 uM, p<0.05, n=5) whereas P1 Glade C failed to induce TSLP secretion. P1 Glade C differs from P1 Glade B and A by the ELDKW motif, we have previously shown to be determinant in P1 Glade B binding to Galactosyl Ceramide (GalCer), the epithelial HIV-1 receptor (Bomsel 1997, Alfsen and Bomsel 2002). We thus hypothesized that P1 Glade B and A interaction with GalCer initiated TSLP secretion. Accordingly, P1 Glade B mutated in W666G (P1W) that fails to interact with GalCer (Alfsen and Bomsel 2002) completely looses the capacity to induce TSLP secretion. Furthermore, when the interaction between P1 and GalCer was blocked by pre-incubation with anti-GalCer antibody, TSLP production is entirely blocked, confirming that TSLP secretion is initiated by P1 interaction with GalCer (FIG. 1C). Importantly, P1 stimulation also induces primary human nasal epithelial cells (HNECs) to secrete TSLP upon a GalCer-dependent manner (FIG. 1D).

[0125] Long Form TSLP is Up-Regulated after P1 Stimulation

[0126] Two transcript variants of TSLP, namely the short (sfTSLP) and the long (lfTSLP) forms, were recently identified (Tsilingiri, Fornasa et al. 2017). The expression of sfTSLP has been suggested to be constitutive and homeostatic whereas the lfTSLP leads to proinflammatory responses (Tsilingiri, Fornasa et al. 2017). We thus investigated which form(s) of TSLP was upregulated by P1 stimulation of nasal epithelial cells. When analyzed at the transcriptional level in nasal RPMI and primary HNEC cells, the expression of sfTSLP and lfTSLP differs by a factor >102. Upon P1 stimulation of both nasal RPMI cells and primary HNECs, the level of the sfTSLP transcript remains unchanged (data not shown). In contrast upon P1 stimulation, the level of lfTSLP transcription in both nasal RPMI cells and HNECs increased by 1.9 (p=0.02, n=5) and 5.9-fold (p=0.004, n=6), respectively, compared to unstimulated cells. Altogether, these results indicate that P1 upregulates lfTSLP selectively at a transcription level.

[0127] P1-Induced lfTSLP Expression is Regulated by miR-4485, Calcineurin and PAR-2

[0128] Next, we investigated the intracellular mechanisms leading to TSLP expression after P1 interaction with GalCer. We concentrated on primary HNECs as its increase in lfTSLP transcription level upon P1 stimulation is higher compared to that in nasal RPMI cells (data not shown).

[0129] We have previously shown that the non-coding microRNA miR-375 controls TSLP expression in primary human foreskin keratinocytes (Zhou, Xu et al. 2018), as it does in human intestinal cell lines (Biton, Levin et al. 2011). When tested in primary HNECs, we found that the TSLP secretion induced by P1 described above is not accompanied by a change in miR-375 expression. We thus further investigated the microRNAs profiles upon nasal epithelial HNECs stimulation by P1 after treatment with or in absence of P1 for 6 hours, comparatively by microRNA array analysis. As a result, 39 microRNAs are differentially expressed with a fold change ranging from 1.3 to 9.15 when p<0.05, including 23 up-regulated and 16 down-regulated genes (data not shown).

[0130] Remarkably, in the microRNA array analysis, the highest upregulated gene upon P1 stimulation is the miR-4485-3p with a >9-fold increase (data not shown). We validated this up-regulation by qPCR resulting in an increase in miR-4485-3p expression by 2.60.8-fold (n=4) upon P1 stimulation (data not shown). MiR-4485-3p is a relatively newly described microRNA that is poorly characterized at the experimental level. The only described activity of miR-4485-3p is to regulate mitochondrial functions suggesting a role in tumor suppression (Sripada, Singh et al. 2017). Thus, we first evaluated whether this microRNA controlled TSLP expression. Therefore, primary HNECs were transfected with a specific siRNA to inhibit miR-4485-3p expression before P1 stimulation. As a result, knocking down miR-4485-3p by 50-60% decreases, in turn, P1-induced TSLP expression by 4810% (p<0.01, n=4), compared to cells transfected with a mock inhibitor (data not shown).

[0131] Bioinformatics analyses were conducted to further elucidate the mechanisms by which P1 modulates all identified microRNAs and subsequent intracellular signaling pathways. The genes predicted to be targeted by identified microRNAs participate in several signaling pathways, the five principals including G protein-coupled receptor (GPCR) associated signaling, Nuclear factor of activated T-cells (NFAT) signaling, Rho GDP signaling, Ephrin receptor signaling, and thrombin signaling (data not shown).

[0132] Corroborating this predictive analysis designating NFAT, GPCR, and thrombin (PAR associated) pathways (Coughlin 2000) as the main ones induced by P1 stimulation, it has been described that in keratinocytes, TSLP production is regulated by Ca2+-dependent NFAT signaling itself triggered by the activation of GPCR protease-activated receptor 2 (PAR-2) (Wilson, The et al. 2013). Thus, we next evaluated experimentally whether inhibitors specific to these pathways also reduced P1-induced TSLP expression in primary HNECs. Therefore, HNECs were pre-treated with the calcineurin inhibitor Cyclosporine A (CsA), or with the PAR-2 antagonist ENMD-1068 prior to P1 stimulation. Accordingly, TSLP expression was reduced by 674% (p<0.001, n=3) upon CsA pre-treatment and by 4624% (p<0.05, n=3) following ENMD-1068 pre-treatment (data not shown). Furthermore, CsA and ENMD-1068 inhibitors also blocked the up-regulation of miR-4485-3p (data not shown). In contrast, blocking NF-B and MAPK with Dexamethasone (Dex) had no effect on TSLP expression (data not shown). These results provide direct and indirect evidence that miR-4485-3p, calcineurin, and PAR-2 mediated signaling tightly correlate with P1-induced TSLP expression.

[0133] To further confirm that P1 activates calcineurin, we investigated whether, in nasal epithelial cells, P1 induces calcium fluxes that generally causes calcineurin activation (Hogan, Chen et al. 2003). Accordingly, using fluorescent dye Fura-2/AM imaging technology, we observed in both nasal RPMI cells and primary HNEC cells, that P1 treatment induces an immediate extracellular calcium influx in a concentration-dependent manner (125 M vs 2511M of P1, n=3) (data not shown). In contrast, treatment with control peptides (P1-5W mutant and P1 Glade C, both at 125 M) fails to raise the calcium level significantly.

[0134] Together, these data indicated that in nasal epithelial cells, P1-stimulated TSLP expression is regulated by miR-4485 via a Ca2+-dependent NFAT signaling pathway through the interaction with PAR-2 receptor.

[0135] P1 Further Stimulates Epithelial Secretion of MMP-9, CCL20, CCL2, and IL-10

[0136] We next investigated whether, in addition to TSLP, P1 could stimulate epithelial secretion of additional immune factors prone to attract antigen presenting cells (APCs). Therefore, nasal RPMI cells where incubated with P1 (125 M) and after 24 hrs, the cell culture medium was analyzed for interleukin (IL)-25/IL-17E, IL-33, IFN-, IL-10, IL-12/23p40, IL-4, IL-5, IL-6, IL-13, TNF-, Matrix metalloproteinase 9 ((MMP-9), IL-8/CXCL8, MIP-3/CCL2, MCP-1/CCL20, MDC/CCL22, TARC/CCL17, APRIL and BAFF by Luminex technology. As a result, P1 selectively induced the secretion of MMP-9, CCL20, CCL2 and IL-10 (data not shown). Furthermore, as observed for P1-induced TSLP secretion, P1W and P1 Glade C were unable to stimulate significant MMP-9, CCL-20 CCL2 or IL-10 production. Together with TSLP (Zhou, Xu et al. 2018), this set of immune factors could facilitate recruitment of APCs to the mucosal surface for initiation of an immune response, since CCL20 and CCL2 chemo-attract macrophages and immature dendritic cells (DCs), and MMP-9 degrades the extracellular matrix and facilitates the migration of immune cells in or out the epithelium. Treg cells have IgA-inducing functions and require RA, TGF-b1, IL-10, and TSLP from intestinal epithelial cells and DCs. So, we assumed IL-10 released from either EC or DC may contribute to IgA class switching (Gutzeit, Magri, et al. 2014).

[0137] P1 Activates Human Dendritic Cells in a Nasal Mucosal Model

[0138] APCs link the innate and adaptive immune systems and determine the polarization of the immune responses. APCs are thus a key target in vaccine and adjuvant development (Coffman, Sher et al. 2010). DCs being the most abundant APCs in airway mucosa (Schon-Hegrad, Oliver et al. 1991), we further investigated mucosal DCs responses to P1 stimulation.

[0139] It has been suggested that mucosal DCs display unique functions due to the local microenvironment, especially at mucosal level (Brandtzaeg 2009). In particular, mucosal DCs modulate their functions by interacting with epithelial cells (ECs) including via epithelial secretion of TSLP (Rimoldi, Chieppa et al. 2005, Biton, Levin et al. 2011). Thus, we established a simplified mucosal DC model, by co-culturing DCs and nasal ECs (RPMI-2650 cell line), thereby mimicking the nasal mucosal environment. DCs were first educated by a 24 hr co-culture with ECs. Subsequently, these educated DCs were either maintained in culture with ECs and referred to as DC-EC, or separated from the epithelium and referred to as eduDC. Alternatively, DCs only cultured with medium represented non-mucosal DCs.

[0140] Each type of DCs was stimulated with P1 overnight and the expression of maturation markers was assessed by flow cytometry. Compared to untreated cells, P1-treated mucosal DCs, either DC-EC or eduDCs, show a significant up-regulation of co-stimulatory molecules CD83 (data not shown) and CD86 (data not shown). In contrast, P1 has no effect on non-mucosal DCs. Surprisingly, P1 also significantly enhanced the expression of TSLP receptor, with both chain TSLP-R (data not shown) and IL-7R (data not shown) being upregulated on the DCs in all three models.

[0141] The cytokine and chemokine secretion profile were also studied in these models, comparatively. Compared with non-mucosal DCs, P1 induces a significant increase in IL-6, IL-8, IL-10, CCL20, CCL22 and MMP-9 secretion in eduDC and DC-EC models as well as that of TSLP secretion, although more modest. In contrast, IFN- secretion remains unchanged upon P1-stimulation or slightly decreases in DC-EC (data not shown). In addition, several cytokines, such as IL-25, IL-33, IL-4, IL-5, remain undetectable whatever the model, whereas others, such as IL-12, IL-13, CCL2, CCL17, TNF-, APRIL and BAFF, are secreted equally in all three models.

[0142] Activated DCs are known to stimulate T-cell proliferation to initiate an adaptive immune response, both in vivo and in vitro (Fontenot, He et al. 2009, Yeh, Yeh et al. 2013, Qin, Yin et al. 2015). Therefore, we further assessed if P1 activated mucosal DCs could promote T cell proliferation. As a result, P1 primed eduDCs induced the proliferation of autologous CD4+ T cells, whereas treatment with control peptides (P1W mutant and P1 Glade C) or P1 stimulation on CD4+ T cells alone has no effect (data not shown). Similar results were observed with DC-ECs, in agreement with the similar cytokine profiles between DC-EC and eduDC as described above.

[0143] Altogether, these results show that P1 activates mucosal DCs specifically, resulting in Th2 cytokine and chemokine secretion, and in CD4+ T cell proliferation.

[0144] P1 Acts as an Adjuvant to Stimulate Antigen-Specific Humoral Responses In Vitro

[0145] Finally, given that P1 induces various immuno-modulatory effect in mucosal cells involved in vaccination at the nasal site, as described above, we investigated whether P1 was able to act as an adjuvant. Using an in vitro immunization model with human PBMCs, the capacity of P1 to trigger a humoral immune response against a well-characterized antigen, namely ovalbumin (OVA), was evaluated.

[0146] In vitro immunization assays have been used to produce specific monoclonal antibodies using a defined antigen complemented with an adjuvant (Borrebaeck, Danielsson et al. 1987). Here, we establish a mucosal immunization model adapted from (Jung, Matsumoto et al. 2007, Yeh, Yeh et al. 2013, Wijkhuisen, Savatier et al. 2016) and using mucosal DCs based on our results presented above. OVA was selected as the model antigen. Therefore, human CD8-depleted PBMCs (n=5 independent donors) were cocultured with RPMI 2650 cells for one day to educate DCs, and prior to addition of either medium, OVA, OVA plus P1 mutant (P1mut, 125 M) or OVA plus P1 (5 M, 25 M, 125 M) for seven more days. OVA-specific B cells were quantified by flow cytometry using FITC-conjugated OVA and anti-CD2O-PE to gate on OVA-specific B cells. The Ig isotype of surface B cell receptor (BCR) was next characterized by APC-conjugated anti-human-IgA or anti-human-IgG. As shown in FIG. 2 (A, B and C), OVA alone, similarly to medium, failed to induce OVA-specific specific B cells, whereas in the presence of P1, OVA-specific B cells were detected. At 5 M and 25 M, the concentration at which P1 remains in the monomeric state, induction of OVA-specific B cells is very limited, whereas, at 125 M, P1 significantly enhances OVA recognition by B cells due to surface expression of OVA-specific IgA and IgG isotypes. Similar results were obtained when B cells were stained with anti-CD19-PE. Importantly, in the absence of nasal epithelial cells during the in vitro immunization, P1 is not able to induce OVA-specific B cells. Within the culture supernatants of day 7, OVA-specific antibody secretion could not be detected by ELISA, most likely because blasts were not formed at this early time point of the immunization and in agreement with the detection of OVA-specific IgA and IgG at the B cell surface, prior to blast differentiation. Hence, P1 appears to act as an adjuvant by promoting the expression of antigen-specific BCR on B cells, which may need additional signals to develop into plasma cells.

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