Cutting Edge: A Dual TLR2 and TLR7 Ligand Induces Highly Potent Humoral and Cell-Mediated Immune Responses

This information is current as Alice Gutjahr, Laura Papagno, Francesco Nicoli, Alain of September 25, 2021. Lamoureux, Fabienne Vernejoul, Thierry Lioux, Emma Gostick, David A. Price, Gérard Tiraby, Eric Perouzel, Victor Appay, Bernard Verrier and Stéphane Paul J Immunol published online 21 April 2017

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 21, 2017, doi:10.4049/jimmunol.1602131 Th eJournal of Cutting Edge Immunology

Cutting Edge: A Dual TLR2 and TLR7 Ligand Induces Highly Potent Humoral and Cell-Mediated Immune Responses ,†,‡ x,{ x,{ Alice Gutjahr,* Laura Papagno, Francesco Nicoli,‖ Alain Lamoureux,*‖ Fabienne Vernejoul,* Thierry Lioux,* Emma Gostick,x { David A. Price, Ge´rard Tiraby,* Eric Perouzel,* Victor Appay, , ,# Bernard Verrier,† and Ste´phane Paul‡ TLR agonists are currently being developed and tested crosstalk between TLRs has been in the spotlight recently (4, as adjuvants in various formulations to optimize the im- 5), together with the adjuvant potential of covalently linked munogenicity and efficacy of vaccines. The aim of this pattern recognition ligands (6). TLR2 is involved in Downloaded from study was to evaluate the immunostimulatory properties the recognition of a wide array of cell wall components, such of a novel compound incorporating covalently linked as peptidoglycan. Although stimulation of TLR2 by the moieties designed to stimulate both TLR2 and TLR7. synthetic ligand Pam3Cys induces a TH2-biased response (7), This dual TLR2/TLR7 agonist induced the maturation cellular activation via the same pathway can also promote of dendritic cells and primed substantial populations TH1-mediated (8). TLR7 plays an important role of cytolytic and highly polyfunctional effector CD8+ in the immune response to viral infection by recognizing http://www.jimmunol.org/ ssRNAs. Stimulation of TLR7 induces strong TH1-polarized T cells in vitro, and safely potentiated the immunoge- + nic properties of a nanoparticulate Ag in vivo, eliciting immunity (9) and has been shown to enhance CD8 T cell responses in nonhuman primates (10). humoral responses with a balanced T 1/T 2profilein H H In this study, we evaluated the immunostimulatory prop- mice. Collectively, these data reveal the potential utility of erties of a novel chimeric compound designed to stimulate both chimeric adjuvants with synergistic activities mediated via TLR2 and TLR7. Several approaches were used to characterize TLRs. The Journal of Immunology, 2017, 198: 000–000. the adjuvant effects of this dual ligand in vitro, including assays

of function, (DC) maturation, and by guest on September 25, 2021 + accination is one of the major successes of medicine. effector CD8 T cell priming (11). Safety and efficacy were However, infectious diseases continue to cause sub- also assessed in vivo, using a mouse model of vaccination with V stantial morbidity and mortality worldwide. Although nanoparticulate HIV-1 p24. most current vaccines operate via the induction of long-lived humoral immunity, cellular immune responses will likely be Materials and Methods critical for protection against intracellular pathogens (1). More- TLR ligands and HIV-1 p24 over, the advantages of recombinant and synthetic PamadiFectin is a chimeric molecule incorporating the TLR7 agonist CL307 peptides as vaccine Ags with respect to safety and production covalently linked via a spermine group to the TLR2 agonist Pam2C designed to stimulate both TLR2 and TLR7 (tlrl-c553; InvivoGen) (Supplemental Fig. are frequently offset by poor immunogenicity (2). Potent new 1A). HIV-1 p24 Ag was produced and purified by PX’Therapeutics. adjuvants are therefore required to facilitate the development of more effective vaccines. Determination of ligand-specific activity TLRs have distinct functions in the HEK-Blue human (h)TLR2 and hTLR7 reporter cell lines (InvivoGen) were and are commonly used as vaccine adjuvants (3). Synergistic used as specified by the manufacturer to detect TLR2 and TLR7 stimulation.

*InvivoGen, 31400 Toulouse, France; †Laboratoire de Biologie Tissulaire et d’Inge´nierie Investigator. B.V. and S.P. are supported by Fondation Pierre Berge´ (Sidaction); Har- The´rapeutique, UMR5305, Universite´ Lyon 1, CNRS, 69007 Lyon, France; ‡Groupe monizing, Integrating, and Vitalizing European Research on AIDS/HIV (Rationally Immunite´ des Muqueuses et Agents Pathoge`nes, Faculte´ de Me´decine de Saint-Etienne, Designed Therapeutic Vaccine against HIV-1 Based on a Novel Formulation of INSERM Centre d’Investigation Clinique en Vaccinologie 1408, 42023 Saint-Etienne, Nanoparticle-Protected mRNA); and by European Union FP7 Grants Cutaneous x France; Sorbonne Universite´s, Universite´ Pierre et Marie Curie – Universite´ Paris VI, and Mucosal HIV Vaccination (241904) and Advanced Immunization Technologies Departement Hospitalo-Universitaire “Vieillissement Immunitaire et Stress,” Centre (280873). { d’Immunologie et des Maladies Infectieuses, 75252 Paris, France; INSERM U1135, ‖ Address correspondence and reprint requests to Ste´phane Paul, Faculte´ de Me´decine de Centre d’Immunologie et des Maladies Infectieuses, 75252 Paris, France; Institute of Saint-Etienne, INSERM Centre d’Investigation Clinique en Vaccinologie 1408, 15 rue Infection and Immunity, Cardiff University School of Medicine, Cardiff CF14 4XN, Ambroise Pare´, 42023 Saint-Etienne, France. E-mail address: [email protected] United Kingdom; and #International Research Center of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan The online version of this article contains supplemental material.

ORCIDs: 0000-0001-8084-2125 (L.P.); 0000-0001-6327-5958 (F.N.); 0000-0002- Abbreviations used in this article: C-H2DCFDA, 5(6)-carboxy-29,79-dichlorodihydro- 0068-4516 (E.P.); 0000-0002-8478-7095 (B.V.). fluorescein diacetate; DC, dendritic cell; h, human; moDC, monocyte-derived DC; NP, nanoparticle; PLA, polylactic acid; ROS, reactive oxygen species. Received for publication December 23, 2016. Accepted for publication March 28, 2017. A.G. is supported by an Association Nationale de la Recherche et de la Technologie Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 doctoral fellowship in collaboration with InvivoGen. D.A.P. is a Wellcome Trust Senior

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602131 2 CUTTING EDGE: A NEW CHIMERIC ADJUVANT TLR2/TLR7

In vitro maturation of human monocyte-derived DCs In vivo immunogenicity of vaccine formulations PBMCs were isolated from venous blood, and monocytes were purified and Six-week-old female BALB/c mice (Charles River Laboratories) were hosted at differentiated into monocyte-derived DCs (moDCs) as described previously the Plateau de Biologie Expe´rimentale de la Souris of the Ecole Normale (12). After 6 d, different concentrations of TLR2, TLR7, or chimeric TLR2/ Supe´rieure in Lyon. All studies and procedures were conducted in accordance TLR7 ligands were added to the moDCs, and the cultures were incubated for with institutional guidelines and approved by the Comite´ Rhoˆne-Alpes a further 24 h. Cells were then stained with anti–CD1a-FITC, anti–CD80- d’Ethique pour l’Experimentation Animale (Lyon, France). Mice were di- PE, and anti–CD86-PE mAbs (BD Pharmingen). Data were acquired using a vided into seven groups of five animals and immunized s.c. on days 0, 14, and FACSCanto flow cytometer (BD Biosciences) and analyzed with FlowJo 28 with 10 mg of p24, either adsorbed on PLA NPs (NP-p24) or coad- software (Tree Star). RNA was extracted from moDCs using an RNeasy Mini ministered with AddaVax (an oil-in-water emulsion with a formulation Kit (Qiagen), and the expression of 84 was quantified using an RT2 similar to MF59 adjuvant; InvivoGen), suspended in a final volume of 100 ml Profiler PCR Arrays hTLR Signaling Pathway Kit (Qiagen). of PBS. In the adjuvanted groups, a total of 20 nmol ligand (TLR2, TLR7, both or chimeric, encapsulated or not) was coadministered with PLA-p24. Measurement of reactive oxygen and nitrogen species One group was immunized with NP-p24 alone. Sera, feces, and vaginal washes were collected on days 0, 14, 28, and 42 and treated as described The intracellular accumulation of reactive oxygen species (ROS) was measured previously for subsequent ELISA-based analyses (14). Affinity assays were after staining with 5(6)-carboxy-29,79-dichlorodihydrofluorescein diacetate performed by ELISA as described by Mann et al. (15). Serum CXCL13 levels (C-H2DCFDA; Life Technologies). Murine RAW 264.7 were were determined in 10-fold diluted samples using a DuoSet ELISA Mouse cultured in 12-well plates and stimulated with different ligands for 16 h in CXCL13 Kit (R&D Systems). ELISPOT assays were performed with mouse DMEM supplemented with 5% FCS. After incubation, cells were stained for splenocytes as described previously (16). 30 min with 5 mM C-H2DCFDA and analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed with Statistical analysis FlowJo software (Tree Star). Downloaded from The production of NO was measured indirectly via the stable metabolite Statistical analyses were performed using GraphPad Prism software. All values nitrite using Griess reagent (Promega). Murine RAW 264.7 macrophages were and error bars represent mean 6 SD. cultured in 24-well plates and stimulated with different ligands for 24 h. Nitrite concentrations were calculated against a standard curve to quantify NO. Results and Discussion In vitro priming of effector CD8+ T cells PamadiFectin induces moDC maturation in vitro The in vitro priming assay was performed as described previously (11). Briefly, After demonstrating that PamadiFectin stimulated both TLR2 PBMCs from healthy HLA-A2+ individuals were supplemented with FLT3L and TLR7 (Supplemental Fig. 1B), we analyzed its ability to http://www.jimmunol.org/ 3 6 (50 ng/ml; R&D Systems) and cultured at 5 10 cells per well in a 24-well induce DC maturation. To do so, we measured ligand- plate. After 24 h, maturation of DCs was induced with different ligands or a standard mixture, added together with the ELA-20 peptide specific upregulation of the differentiation markers CD80 ELAGIGILTV (YTAAE ILGVL; Melan-A/MART-1 residues 21–40A27L)ata and CD86 on human moDCs generated in vitro. In contrast + final concentration of 1 mM. ELA-specific CD8 T cell frequency and pheno- to the TLR2 and/or TLR7 ligands at 1 mM, PamadiFectin at type were determined on day 10. Data were acquired using an LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). the same concentration induced significant expression of CD80 and CD86 (Fig. 1A). We further analyzed the expres- Preparation of polylactic acid nanoparticles sion of 84 genes associated with the TLR signaling pathway in by guest on September 25, 2021 Empty and PamadiFectin-loaded nanoparticles (NPs) were prepared by moDCs after stimulation with PamadiFectin or a combina- nanoprecipitation as described previously (12, 13). Briefly, the polymer was tion of TLR2 and TLR7 ligands (Fig. 1B). These data confirmed dissolved in acetone with or without PamadiFectin and added gradually to an the upregulation of CD80 and showed that PamadiFectin in- aqueous solution (ethanol/water) under slow stirring. Organic solvents were then removed under reduced pressure at 30˚C. Particle size, polydispersity, duces an overexpression of genes encoding proinflammatory and surface charge were determined at 25˚C using a Zetasizer Nano ZSP associated with both TH1(IFN-g,IL-2)andTH2 (Malvern). The TLR2/TLR7 ligand encapsulation rate was determined after (IL-6, IL-10) polarization. Unlike the coadministration of centrifugation of the NP solution. Free ligand remaining in the supernatant TLR2 and TLR7 ligands, PamadiFectin also induced over- was cultured with HEK-Blue hTLR2 and hTLR7 reporter cell lines (Invi- voGen). The p24-coated polylactic acid (PLA) NPs were elaborated as de- expression of the adaptor MyD88 and transcription scribed previously (12). factors such as IFN regulatory factor 1 and REL. In line with

FIGURE 1. PamadiFectin is a potent immu- nostimulant. (A) Expression of the costimulatory molecules CD80 and CD86 on the surface of CD1a+CD14– moDCs generated in vitro was de- termined by flow cytometry 24 h after stimulation with the indicated ligands at a concentration of 1 mM. (B) Expression profile of TLR pathway genes (gray, no change in expression; white, down- regulation .5-fold; light gray, downregulation .2-fold; dark gray, upregulation .2-fold; black, upregulation .5-fold). (C)MurineRAW264.7 macrophages were cultured in the presence of the indicated ligands. ROS were stained with C-H2DCFDA and analyzed by flow cytometry. Nitrite was revealed with Griess reagent and quantified using a plate reader. The data represent three independent experiments conducted in du- plicate. Statistical significance between two groups was determined using an uncorrected Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 2. PamadiFectin primes CD8+ T cells with cytolytic potential in vitro. PBMCs from healthy HLA-A2+ individuals were stimulated with FLT3L and primed with the Melan-A/MART-1 epitope ELA for 10 d in the presence or absence of the indicated ligands at a concentration of 2.5 mM. (A and B) Tetramer- binding CD3+CD8+ cells and intracellular expression of granzyme B and T-bet were quantified by flow cytometry. Statistical significance between two groups was by guest on September 25, 2021 determined using an uncorrected Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. ns, p . 0.05. these findings, previous work has shown that costimulation of TNF, and IL-6 (data not shown). Macrophage activity is TLR7-treated moDCs with a TLR2 agonist does not enhance therefore induced efficiently by the dual TLR2/TLR7 ago- maturation (17). Covalent linkage of the individual TLR2 nist PamadiFectin. and TLR7 ligands may therefore be essential for the observed potency of PamadiFectin. The synergistic effect could be PamadiFectin primes CD8+ T cells in vitro explained by endosomal internalization of the TLR7 agonist. Next, we assessed the ability of PamadiFectin to prime Ag- Accordingly, whereas TLR2 is directly accessible on the cell specific naive T cells. In this assay, PBMCs from healthy surface, TLR7 is only expressed within endosomes and re- HLA-A2+ individuals were stimulated with FLT3L and primed quiresagonistengulfmentforintracellular activation. The with the Melan-A/MART-1 epitope ELA in the presence or lipidic TLR2 agonist domain may facilitate the endosomal absence of TLR ligands at a concentration of 2.5 mM. The dual acquisition of PamadiFectin via lipid rafts (18), enabling agonist primed a substantial population of ELA-specific CD8+ direct in situ stimulation of TLR7. Moreover, the chimeric T cells, at least equivalent in magnitude to that elicited by a ∼ agonist can form spontaneous NPs of 50 nm (data not standard mixture of proinflammatory cytokines (Fig. 2A). Of shown). As the nanoparticulate format is crucial for molec- greater note, Ag-specific CD8+ T cells primed in the presence ular adjuvant uptake, this property may further enhance the of PamadiFectin expressed higher levels of granzyme B com- efficacy of PamadiFectin (19, 20). pared with either the cytokine mixture or a combination of the individual TLR2 and TLR7 agonists, indicating more potent PamadiFectin activates macrophages in vitro cytolytic activity (Fig. 2). A similar pattern was observed with ROS and NO are key mediators of innate immunity (21). In the transcription factor T-bet, which regulates TH1 cells and particular, macrophages use both ROS and NO as microbicidal the synthesis of IFN-g and granzyme B in CD8+ T cells (22). molecules in phagolysosomes to kill pathogens. PamadiFectin Overall, the effect of PamadiFectin in this assay was similar to induced the production of ROS in murine RAW 264.7 mac- that of the TLR8 agonist ssRNA40, previously shown to be rophages far more efficiently compared with the TLR2 and/or a potent inducer of effector CD8+ T cells (11). In keeping TLR7 ligands at a concentration of 1 mM (Fig. 1C). The dual with this comparison, the Ag-specific cells primed by the dual agonist also stimulated NO expression, although this effect TLR2/TLR7 agonist were highly polyfunctional, expressing was likely due primarily to stimulation via TLR2. Moreover, TNF, MIP-1b,andIFN-g (SupplementalFig.1C).These PamadiFectin is able to induce the secretion of type 1 IFN, findings suggest that PamadiFectin may be especially useful as 4 CUTTING EDGE: A NEW CHIMERIC ADJUVANT TLR2/TLR7 an adjuvant in the setting of therapeutic vaccination, where improved immune responses relative to NP-p24 alone (5-fold the induction of cytotoxic responses is paramount in the fight increase, p , 0.01), NP-p24 plus CL307 (8-fold increase, p , against cancer and chronic infections (23). 0.001), and NP-p24 coadministered with both TLR2 and TLR7 ligands (3.2-fold increase, p , 0.01). However, the inclusion of PamadiFectin enhances in vivo immune responses against HIV-1 p24 the dual TLR2/TLR7 agonist within the nanoformulation did There is much interest in the potential utility of PLA NPs as Ag not significantly enhance humoral responses compared with carrier systems encapsulating molecular adjuvants (12). To test coadministration of the free molecule, suggesting that the effect this modular delivery system, we coated PLA NPs of ∼250 peaks after a single injection. nm with HIV-1 p24 at a final concentration of 6.7 mg/g PLA. The mucosal immune response was analyzed in vaginal PamadiFectin was encapsulated within the PLA NPs at a loading washes and feces (Supplemental Fig. 1D). Although the dual rate of 0.8% with almost 100% efficiency (NP [PamadiFectin- TLR2/TLR7 agonist did not yield IgA secretion, significantly p24]). The immunostimulatory potential of the encapsulated higher levels of IgG were present in both vaginal lavages and agonist was then tested in mice relative to the free agonist feces retrieved from mice receiving NP-p24 with Pamadi- (NP-p24 1 PamadiFectin) and the individual TLR2 and Fectin relative to formulations without the molecular adju- TLR7 ligands. vant. This effect may be due to the induction of high IgG Coadministration of NP-p24 with the dual TLR2/TLR7 titers in sera, leading to transudation and/or FcRn transport agonist induced high Ab titers 2 wk after the first injection to mucosal surfaces (24). relative to formulations without the molecular adjuvant, al- Systemic toxicity was observed with the individual TLR7 Downloaded from though this difference did not reach statistical significance due (p , 0.01) and TLR2 (p , 0.001) agonists, and coadmin- to the low numbers of mice per group (Fig. 3A). The nano- istration was associated with severe weight loss (p , 0.0001). formulation potentiated the immunostimulatory effect of In contrast, the dual TLR2/TLR7 agonist seemed to be much the chimeric ligand, inducing 3.6-fold higher IgG titers safer (Supplemental Fig. 1E). This effect may be explained by compared with the free agonist (p , 0.05) and 20-fold higher thefactthatPamadiFectincontains a lipidic moiety, leading to the IgG titers compared with NP-p24 alone (Fig. 3B, p , 0.001). formation of 50-nm particles, which in turn may limit dissemi- http://www.jimmunol.org/ Moreover, the greater adjuvanticity of this molecule was main- nation and enhance uptake by APCs, thereby avoiding excessive tained after three immunizations, as evidenced by substantially inflammation as recently described for a TLR7 agonist (25). by guest on September 25, 2021

FIGURE 3. Dual TLR2/TLR7 activation enhances humoral responses against HIV-1 p24 in BALB/c mice. Seven groups of mice (n = 5 per group) were immunized s.c. with NP-p24 in the presence or absence of the indicated ligands on days 0, 14, and 28. (A and B) Serum p24-specific IgG titers were measured by ELISA on day 14 (A)orday42(B). (C) Ab avidities (left) and serum CXCL13 concentrations (right) were measured by ELISA on day 42. (D) IgG1 and IgG2a subtypes were measured by ELISA on day 42. (E)IFN-g–secreting cells in mouse splenocytes were counted by ELISPOT. Statistical significance between two groups was determined using an uncorrected Student t test. *p , 0.05, **p , 0.01, ***p , 0.001, ****p # 0.0001. ns, p . 0.05. The Journal of Immunology 5

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Blumberg. 2010. Neonatal Fc receptor: from immunity to therapeutics. J. Clin. Immunol. 30: 777–789. 25. Lynn, G. M., R. Laga, P. A. Darrah, A. S. Ishizuka, A. J. Balaci, A. E. Dulcey, M. Pechar, R. Pola, M. Y. Gerner, A. Yamamoto, et al. 2015. In vivo character- Disclosures ization of the physicochemical properties of polymer-linked TLR agonists that The authors have no financial conflicts of interest. enhance vaccine immunogenicity. Nat. Biotechnol. 33: 1201–1210. 26. Victora, G. D., and M. C. Nussenzweig. 2012. Germinal centers. Annu. Rev. Immunol. 30: 429–457. 27. Havenar-Daughton, C., M. Lindqvist, A. Heit, J. E. Wu, S. M. Reiss, K. Kendric, References S. Be´langer, S. P. Kasturi, E. Landais, R. S. Akondy, et al; IAVI Protocol C Principal 1. Coffman, R. L., A. Sher, and R. A. Seder. 2010. Vaccine adjuvants: putting innate Investigators. 2016. CXCL13 is a plasma biomarker of germinal center activity. immunity to work. Immunity 33: 492–503. Proc. Natl. Acad. Sci. USA 113: 2702–2707. A B

C

PamadiFectin primed

Unstim

+ peptide

D IgG IgA E

washes Vaginal Vaginal

F IFN-γ Feces

Supplementary figure 1: (A) Chemical structure of PamadiFectin. (B) In vitro assessment of ligand-specific activation via the TLR2 and TLR7 pathways in HEK-Blue hTLR2 and hTLR7 cells. (C) PBMCs from healthy HLA-A2+ individuals were stimulated with FLT3L and primed with the Melan-A/MART-1 epitope ELA for 10 days in the presence or absence of the indicated ligands at a concentration of 2.5 µM. Polyfunctionnality of the primed antigen-specific cells was assessed by measuring the expression of CD107a, TNF, IL-2, MIP-1β, IL-2 and IFNγ. (D) Vaginal secretions and feces were collected from mice and p24-specific, diluted respectively to 1/10 and 1/2,and IgA and IgG levels were measured by ELISA on day 42. (E) Mice were weighted before and 24 hr after administration of the vaccine formulations. (F) Serum IFN-γ and IL-5 levels were analyzed 16 hr after the first vaccination by Luminex using the Bio-Plex Pro assay (Bio-Rad). Statistical significance between two groups was determined using an uncorrected Student’s t-test: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P ≤ 0.0001.