The geometry of a TLR2-agonist-based adjuvant can affect the

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The geometry of a TLR2-agonist-based adjuvant can affect the resulting antigen-specific immune response Acep R Wijayadikusumah, Weiguang Zeng, Hayley A McQuilten, Chinn Yi Wong, David C. Jackson, and Brendon Y. Chua Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00026 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Molecular Pharmaceutics

The geometry of a TLR2-agonist-based adjuvant can affect the resulting antigen-specific immune response Acep R. Wijayadikusumah1,2, Weiguang Zeng1, Hayley A. McQuilten1, Chinn Yi Wong1, David C. Jackson1 & Brendon Y. Chua1*

1Department

of Microbiology and Immunology, The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Melbourne, Victoria 3010, Australia

2Research

and Development Division, PT. Bio Farma (Persero), 28 Pasteur St, Bandung, West Java 40161, Indonesia

*Corresponding author. Tel.: +61 3 9035 3129

Email: [email protected]

+ Keywords: dendritic cell, lipopeptide, TLR2, antibody, CD8 T cells

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Targeted delivery of otherwise non-immunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, R4Pam2Cys which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here we compare the immunological properties of ovalbumin (OVA) formulated with different geometrical configurations of R4Pam2Cys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of R4Pam2Cys are more effective at inducing immune responses than are linear geometries. CD8+ T cell-mediated responses are particular improved resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched R4Pam2Cys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signalling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8+ T cells resulting in rapid cell division and upregulation of early cell surface activation markers. These results indicate that as well as cognate recognition of Pam2Cys by TLR2, the adjuvant’s efficiency is also dependent on its geometry.

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INTRODUCTION The discovery that agonists of Toll-like receptors (TLRs) can act as adjuvants has provided opportunities to develop rationally designed interventions against bacterial and viral infections as well as providing immunological solutions against non-infectious diseases and normal physiological conditions. These agonists not only facilitate delivery, uptake and processing of vaccine antigens by antigen presenting cells (APCs), especially dendritic cells (DCs), but concurrently induce the necessary signalling requirements resulting in their activation and migration to draining lymph nodes where antigen-specific humoral and cell-mediated responses are generated 1,2. It is not surprising that optimal induction of immune responses, especially those mediated by CD8+ T cells, are best achieved if both agonist and antigen are associated so that uptake and activation can occur within the same APC

3-7.

The use of peptide-based agonists, notably palmitic acid-based

lipopeptide derivatives, have proven to be very useful for this purpose; the immunogenicity of otherwise non-immunogenic antigens can, for example, be markedly enhanced if linked electrostatically to cationic peptide-based agonists 4,6,8. Compared with the chemistries necessary for covalent attachment, noncovalent association based on the ability of an agonist to electrostatically bind to an antigen provides a simple and straight forward approach to vaccine preparation simplifying manufacturing processes. Our own studies have demonstrated the immunological benefits of non-covalent association of protein antigens with the TLR2 agonist-based cationic lipopeptide R4Pam2Cys

4,8,9.

Its ability to

associate electrostatically with antigen is mediated by a simple two-tiered branched lysine (K) scaffold which bears N-terminal arginine (R) residues in a fan-like arrangement which provides positively charged primary amino groups and guanidinium functional groups for interaction with antigen through multiple electrostatic and hydrogen bonding opportunities under physiological conditions. At the same time the lipid portion, S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys), facilitates delivery of the antigen to the endocytic receptor TLR2 which is expressed on the surface of DCs. Antigen cargo transported into DCs via R4Pam2Cys is processed and presented in the context of MHC molecules and efficiently traffics into the draining lymph nodes to result in effective activation of naïve T cells 8,9. Each vaccine candidate that we have so far assembled using this delivery system has demonstrated significantly enhanced immunogenicity characterised by the induction of robust cytotoxic CD8+ T cell responses as well as strong neutralizing antibody responses when compared to non-adjuvanted antigen 4,9-12. We have also shown that this delivery system is superior to traditional adjuvants at promoting memory T cell development and protective responses against influenza viral infection 9,11. Several studies have highlighted significant differences exhibited by branched and linear 3



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peptide-based immunogens not only in how they are processed and presented by APCs 13-15 but also in the magnitude and type of immune responses that are induced 16-18. In this study, we determined ways in which the biological effects of complexes formed between antigen and R4Pam2Cys are affected by the geometrical arrangement of the component lysine and arginine residues, specifically in branched or linear conformation. We assembled three different structures each containing Pam2Cys but differing in the arrangement of the 4 component R residues. The first of these structures, R4Pam2CysB, contains a branched lysine structure providing attachment points for four N-terminal R residues each of which contribute 2 positive electrical charges thereby imparting an overall charge of 8+ve to the molecule. The second lipopeptide, R4Pam2CysP has a linear K backbone from which the R residues are pendant again providing an overall electrical charge of 8+ve. In the third configuration, R4Pam2CysL four R residues are again present but in this case they are in linear contiguous sequence providing a net electrical charge of 5+ve. In order to characterise and assess the adjuvanting properties of these different configurations of R4Pam2Cys, we examined (i) their size when complexed with the model antigen ovalbumin (OVA), (ii) their in vitro biological effects on dendritic cells (DCs) determined by measuring antigen uptake, cell maturation and cytokine production, (iii) their ability to elicit antibody- and CD8+ T cell-mediated antigen-specific immune responses in vivo, (iv) their efficiency in presenting antigen to T cells in vivo (v), their ability to mediate signal transduction in a TLR2-dependent manner and (vi) their susceptibility to proteolytic degradation.

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MATERIALS & METHODS Synthesis of R4Pam2Cys-based adjuvants The synthesis of R4Pam2CysB was performed as previously described 4, with the modification that the Pam2Cys lipid moiety was attached prior to assembly of the peptide segment 19. Assembly of the variants R4Pam2CysP and R4Pam2CysL used similar acylation reactants and conditions. For R4Pam2CysP (Fig 1A), Dde-Lys(Fmoc)-OH was first coupled to the solid support (PEG-SRAM resin, substitution of 0.24 mmol/g; Rapp Polymere, Germany) in 4-fold molar excess in the presence of equimolar

amounts

of

O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexa-fluoro-phosphate

(HBTU; Protein technology, Inc, USA.), 1-hydroxybenzotriazole (HOBt; CEM, USA), and a 6-fold molar excess of diisopropylethylamine (DIPEA; Sigma Aldrich, Australia) followed by removal of the Fmoc protective group from the C-terminal K and addition of two serine (S) residues followed by Pam2Cys. The Dde group from the C-terminal K was then removed and followed by four rounds of acylation using Fmoc-Lys(Dde)-OH. Removal of the Dde protecting groups from the four contiguous K residues then allowed addition of an R residue at each of the four pendent -amino groups. The Nterminal amino group present on the fifth K residue was acetylated so that the final product had a net electrical charge of +8. For R4Pam2CysL (Fig 3A), four rounds of acylation using Fmoc-Arg(Pbf)OH were carried out once Pam2Cys had been coupled to the -amino group of the C-terminal K residue to yield a lipopeptide containing four consecutive arginine residues in linear configuration. Particle size analysis of lipopeptide and antigen formulations by dynamic light scattering (DLS) The association between OVA and R4Pam2CysB, R4Pam2CysP and R4Pam2CysL was measured using dynamic light scattering (DLS) by mixing 10μg of endotoxin-free ovalbumin (OVA, 0.22 nmol; InvivoGen, California, USA) with a 3-fold molar excess of lipopeptide (0.66nmoles of R4Pam2CysB [1.31µg], R4Pam2CysP [1.42µg] or R4Pam2CysL [1.01µg]) or each lipopeptide alone (0.66nmoles) in 50μL of saline in a microtube. The size distribution and polydispersity of particles were measured in 4μl disposable cyclic olefin co-polymer cuvettes installed in a DynaPro NanoStar DLS instrument (Wyatt Technology, Santa Barbara, USA). The profiles of particle sizes are shown as particle radii (Fig 1D-F) and as the average diameter in nm and the associated percent polydispersity (base-line width of size distribution/mean size distribution). Solutions with particles that are < 20% polydispersed can be considered to be homogeneous 20. Effects of lipopeptide formulations on murine dendritic cells Immature murine dendritic cells, D1 cells 4,21, were derived by culturing splenocytes from a naïve mouse in D1 medium (Iscove’s Modified Dulbecco’s Medium [Gibco, USA] supplemented with 10% fetal calf serum [FCS], 30% fibroblast supernatant [obtained from cultured 3T3 cells] 5



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recombinant murine GM-CSF [50 ng/ml; BioLegend, USA], gentamicin [12 mg/ml], L-glutamine [2mM], sodium pyruvate [1 mM], penicillin [100 IU/ml], streptomycin [100 mg/ ml], and 2mercaptoethanol [55mM]). Fresh D1 medium was replenished every 2-4 days for 21 days and the expression of CD11c and MHC Class II confirmed before use by staining with FITC conjugated antimurine I-A/I-E (Clone M5/114.15.2) and PE-conjugated CD11c antibodies (Clone 2G9; both from Biolegend, USA). DC activation was measured by incubating cells (2×105) with 0.02, 0.2, or 2 ng (0.44, 4.4, or 44fmol) of OVA pre-mixed with a 3-fold molar excess of each lipopeptide (1.33, 13.3 or 133.2fmoles respectively corresponding to 2.6, 26 or 260ng of R4Pam2CysB; 2.8, 28 or 280ng of R4Pam2CysP and 2.1, 21 or 210ng of R4Pam2CysL) in a total volume of 500μL. DCs were also incubated with OVA alone, lipopolysaccharide (LPS; from E.coli O55:B5, Sigma-Aldrich, USA) or maintained untreated. After an incubation period of 14 hours, cells were stained with FITC-anti-I-A/I-E antibody and samples analyzed using a FACS Canto II flow cytometer (BD Biosciences, USA) and data analyzed using FlowJo software (Tree star, Inc, USA). OVA labelled with FITC using a Fluorescein-EX Protein Labeling Kit (Molecular Probes, CA, USA) was used to determine antigen uptake. D1 cells (2×105) were incubated with 5g of FITCOVA alone (0.11nmoles) or formulated with a 3-fold molar excess of lipopeptide (0.33nmoles of R4Pam2CysB [0.65µg], R4Pam2CysP [0.71µg] or R4Pam2CysL [0.51µg]) in a 500L for 3 hours. Cells were harvested 16 hours later, washed and analysed for intracellular fluorescence by flow cytometry following quenching of extracellular fluorescence by incubating cells with an equal volume of 0.1M citrate buffer (pH 4.0) containing 250mg/ml trypan blue (Merck, Damstadt, Germany) for 1 minute on ice 22. Cytokine production was analysed by incubating D1 cells (2×105) with media alone or 1.32fmoles of R4Pam2CysL (0.21µg) or R4Pam2CysB (0.26µg) n 500L and cells harvested after 16 hours and the culture supernatants collected following centrifugation. Cytokine levels were determined using a murine Th1/Th2/Th17 Cytometric Bead Array (CBA) kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Antigen-specific antibody immune responses BALB/c mice (6–8-wk-old) were inoculated by the sub-cutaneous (s.c.) route at the base of tail on day 0 and again on day 21 with 25µg of OVA alone (0.55nmoles) or with OVA formulated with a 3-fold molar excess (1.65nmoles) of lipopeptide (R4Pam2CysB [3.37µg], R4Pam2CysP [3.55µg] or R4Pam2CysL [2.64µg]) in a volume of 100µl. OVA-specific antibodies present in sera obtained 21 and 35 days following inoculation were determined by enzyme-linked immunosorbent assay (ELISA). Mouse antibodies were detected using horse-radish peroxidase-conjugated rabbit anti6


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mouse IgG antibodies (Dako Agilent Pathology Solutions, Denmark) or anti-mouse isotype-specific antibodies (Southern Biotech, Birmingham, USA) for 1 hour followed by the addition of the enzyme– substrate (0.2 mM ABTS in 50 mM citric acid containing 0.004% hydrogen peroxide) and left to develop for 15 min before the addition of 50 mM sodium fluoride to stop the reaction. Antibody titers were expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2. Antigen-specific CD8+ T cell immune responses For the measurement of antigen-specific IFN-γ-production by CD8+ T cells, 6–8-wk-old C57BL/6 mice were inoculated as above. Single cell suspensions were derived from individual spleens obtained 10 days later and resuspended in 5ml of RF-10 media consisting of RPMI-1640 media (Invitrogen, USA) supplemented with 10% inactivated FCS, gentamicin (12mg/ml), 2mM glutamine, 1mM sodium pyruvate, penicillin, streptomycin (100μg/ml) and 55μM 2mercaptoethanol). 100µl of each suspension was then mixed with 100µl of RF-10 media to give a final concentration of 4µg/ml of the H2Kb-restricted epitope OVA257-264 (amino acid sequence SIINFEKL), 5µg/ml GolgiPlug (BD Biosciences, CA, USA) and 10U/ml of recombinant human IL2 (Roche, Mannheim, Germany). Cells were cultured for 6 hours at 37°C. The numbers of IFN-γproducing CD8+ T cells were then determined by intracellular cytokine staining

4

using PerCP-

conjugated anti-murine CD8 (Clone 53-6.7) and APC-conjugated anti-IFN-γ (clone XMG1.2) antibodies (both from BD Biosciences, CA, USA) and analysed by flow cytometry. Antigen-specific CD8+ T cell mediated cytolytic responses were measured in vivo by preparing target cells derived from spleens of naive mice. Splenocytes were stained with 5L Vybant DiD cell-labelling solution (Molecular Probes, Eugene, USA) and pulsed with either: 5g/mL of an irrelevant H-2Kd peptide epitope derived from influenza nucleoprotein (NP147-155; amino acid sequence TYQRTRALV) or the target H-2Kb peptide OVA257-264 (SIINFEKL) at 37oC for 60 minutes. Each cell fraction was labelled with 5-(and 6) carboxyfluoroscein diacetate succinimidyl ester (CFSE; Molecular Probes, USA) at a final concentration of 100nM (CFSElow) or 1M (CFSEhigh). Equal numbers of cells (7.5 × 105 in 200L) from each population were mixed and injected intravenously into mice 7 days after vaccination. Spleens were harvested 16 hours later and examined for the presence of CFSE-labelled OVA257-264-pulsed cells, relative to the number of irrelevant peptidepulsed cells, in each animal by flow cytometry. The in vivo proliferation of T cells induced by vaccination was determined by intravenous transfer of CD45.1+ CD8+ T cells from OT-1 transgenic mice (1×104 cells in 200µl) into naïve C57BL/6 mice 1 day prior to vaccination. Mice were then vaccinated 5µg of OVA alone (0.11nmoles)

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or with OVA formulated with a 3-fold molar excess (0.33nmoles) of lipopeptide (R4Pam2CysB [0.65µg], R4Pam2CysP [0.71µg] or R4Pam2CysL [0.51µg]) in a volume of 100µl Spleens were harvested 7 days later to obtain single cell suspensions and the frequency of CD45.1+ CD8+ T cells measured by flow cytometry following staining with PerCP-conjugated antimurine CD8 and APC-conjugated anti-CD45.1 (clone A20) antibodies (both from BD Biosciences, CA, USA). In vivo antigen presentation CD45.1+ CD8+ T cells (1×107 cells/ml in Hanks Buffered Saline Solution) obtained from OT1 transgenic mice were labelled with 2.5µM CFSE for 10 minutes. Cells were washed extensively with RF-10 media before being transferred intravenously (1×106 cells) into naïve C57BL/6 mice 1 day prior to vaccination. Inguinal lymph nodes were harvested after 72 hours and T cell proliferation measured by flow cytometry to determine the CFSE intensity of CD45.1+CD8+ T cells following staining with fluorochrome-conjugated antibodies. Cells were also stained with PE-Cy7-conjugated anti-CD44 (clone IM7) and BV605-conjugated anti-CD25 (clone 7D4) antibodies. NF-B reporter gene assay for TLR2 signalling HEK293 cells were cultured in a 96-well plate (4×104 cells/well) in a volume of 200µl and transfected transiently 24 hours later with 100ng of an NF-B-luciferase reporter gene, 50ng of a renilla-luciferase expressing plasmid (Promega corporation, USA) with or without 5ng of a TLR2expressing plasmid using the FuGENE method (Roche Diagnostic, Germany). Different amounts of lipopeptide were then added to wells 24 hours later. Cell lysates were prepared 5 hours after stimulation using reporter lysis butter (Promega Corporation, USA) and luciferase activity measured with a reagent kit according to the manufacturer’s instructions (Promega Corporation, USA). TLR2specific NF-B-dependent luciferase activity was normalised by subtracting background activity of samples that were transfected in the absence of TLR2-expressing plasmid. The relative stimulation of NF-B was calculated as the ratio of activity detected in lipopeptide stimulated compared with non-stimulated samples. Trypsin digestion of lipopeptides The susceptibility of each lipopeptide to proteolysis by trypsin was determined by treating R4Pam2CysB, R4Pam2CysP or R4Pam2CysL with trypsin-versene (60μg, Department Microbiology and Immunology, University of Melbourne) in a final volume of 150μL for 10 minutes at 37oC. Solutions were centrifuged at 13,000rpm for 1 minute and 100μL of each supernatant analyzed by reverse phase high pressure liquid chromatography (RP-HPLC) using a Vydac C4 column (6mm x 8


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250mm) installed in a Shimadzu HPLC chromatography system at a flow rate of 2.5ml/min using 0.1% trifluoroacetic acid (TFA) in water and a linear gradient of 0.1% TFA in acetonitrile (VWR Chemicals, USA). Eluted material was detected by monitoring optical density at 214nm; the mass of the molecular species present in eluted material collected at specific times was determined using an Agilent 1100 series LCM/MSD Ion Trap mass spectrometer. Statistical Analyses All statistical analyses were done using Prism 7 software (GraphPad, USA). A Mann Whitney nonparametric T-test was used to determine p-values when the means of only two groups were compared (Figure 4D). All other analyses where the means of more than two groups were determined was done using a one-way ANOVA with a Tukey post-correction test. Asterisks within each figure indicate pvalues of less than 0.05.

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RESULTS Electrostatic association of R4Pam2CysB and R4Pam2CysP with OVA The fan-like arrangement of four R residues in R4Pam2CysB is supported by a K-based scaffold (Fig 1A) which enables it to bind antigen electrostatically facilitating delivery to DCs and inducing strong immune responses 4. We determined how the adjuvanting properties of R4Pam2Cys are affected by arranging the K residues in a linear fashion (Fig 1B; R4Pam2CysP) while allowing it to retain the same electrical charge as R4Pam2CysB. Analysis of the particle sizes formed by R4Pam2CysP, R4Pam2CysB and the model antigen OVA revealed that each are present as polydispersed particles with average sizes 10-fold improvement of these responses compared to OVA alone (Fig 2B). Vaccination of OVA in the presence of either lipopeptide also resulted in similar T cell-mediated cytolytic responses, significantly more OVA257-264 peptide-bearing target cells were killed with each of these two lipopeptide adjuvants than by OVA alone (Fig 2C). Effects of OVA formulated with R4Pam2CysB or R4Pam2CysP on DCs The in vivo activities of these lipopeptides were mirrored by their in vitro effects on DCs. We used cultured spleen-derived CD11c+ D1 cells21 which can be induced activated with LPS to result in the upregulation of surface MHC Class II molecules (Supplementary Figure 1). Incubation of D1 cells with OVA had little or no effect on levels of MHC Class II (Fig 2D). Exposure to OVA formulated with either R4Pam2CysP or R4Pam2CysB, however, induced activation of ~90% cells 10


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which decreased in a dose dependent manner. No significant differences in activation were observed between R4Pam2CysP or R4Pam2CysB at any of the concentrations examined. The ability of each lipopeptide-OVA complex to facilitate antigen uptake was determined by incubating D1 cells with fluorescein isothiocyanate-labelled OVA (FITC-OVA) either alone or when complexed with either lipopeptide. The percentage of cells exhibiting intracellular fluorescence was then determined as a measure of antigen uptake. Antigen was detected in at least 4 times as many cells following exposure to OVA-FITC formulated with R4Pam2CysP or R4Pam2CysB (Fig 2E). Again, there was little or no difference in the levels of antigen uptake mediated by either lipopeptide. Taken together, these results indicate that despite significant differences in geometry R4Pam2CysP and R4Pam2CysB have similar in vivo and in vitro biological properties. Electrostatic association of R4Pam2CysL with OVA and its immunogenicity In the cases of R4Pam2CysB and R4Pam2CysP, the designs allow the primary amino group and the guanidinium group of each R to contribute 2 positive electrical charges each. In order to examine the importance of presenting the R residues in this manner, we examined the biological properties of the molecule R4Pam2CysL in which 4 arginine residues are arranged in a linear fashion i.e. in the absence of any branched structure provided by a scaffold of K residues (Fig 1C). As a consequence of this geometry, the lipopeptide still displays 4 guanidinium groups but has a net positive electrical charge of 5 compared to 8. R4Pam2CysL binds to OVA producing antigen-lipopeptide complexes with a similar size distribution and average diameter as those complexes formed between R4Pam2CysB or R4Pam2CysP and OVA (Fig 1F and Table 1). R4Pam2CysL formulated with OVA elicited significantly higher titres of antibody than OVA alone (Fig 3A) but less antibody than was elicited using R4Pam2CysB. Although there did not appear to be any significant difference in the antibody isotype profiles elicited by each lipopeptide, each formulation elicited IgM antibodies but IgG1 was the most dominant isotype elicited by R4Pam2CysL and R4Pam2CysB with little or no IgG3, IgG2a or IgG2b detected (data not shown). A greater difference between the lipopeptides was observed in their ability to elicit CD8+ T cell responses. The R4Pam2CysB-OVA combination induced significantly more OVA257-264 -specific IFN-γ–secreting CD8+ T cells (Fig 3B) and a stronger cytolytic response against OVA257-264 peptidebearing target cells than did R4Pam2CysL-OVA (Fig 3C). By adoptively transferring OT-1 T cells that recognise the OVA257-264 epitope presented on H2-Kb prior to vaccination, we also showed (Fig. 3D) that the highest frequencies of OT-1 T cells were detected in animals vaccinated with R4Pam2CysB reiterating its advantage over R4Pam2CysL at inducing immune responses.

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Effects of OVA formulated with R4Pam2CysL on DCs and ability to mediate TLR2 signalling Significant differences in the biological activity of R4Pam2CysB and R4Pam2CysL were also apparent when their effects on D1 cells were compared. At all concentrations tested, R4Pam2CysBOVA complexes were more efficient at stimulating and activating DCs than were R4Pam2CysL-OVA complexes (Fig 4A). This difference was also reflected by the ability of DCs to take up R4Pam2CysBFITC-OVA complexes more efficiently than R4Pam2CysL-FITC-OVA complexes (Fig 4B). Exposure of cells to R4Pam2CysB resulted in production of significantly more IL-2, TNF- and IL-6 compared to R4Pam2CysL but neither compound elicited significant amounts of IL-4, IL-10 and IL-17 (Fig 4C). In an attempt to explain the differences in activities of R4Pam2CysB and R4Pam2CysL, we investigated their effects on TLR2-expressing HEK-293T cells using a previously described luciferase-based NF- dependent reporter assay 26. The results (Fig 4D) demonstrate that differences in the levels of signalling between R4Pam2CysB and R4Pam2CysL were apparent at low concentrations with R4Pam2CysB being more effective than R4Pam2CysL. Neither lipopeptide stimulated cells lacking TLR2 (data not shown) confirming TLR2-dependence. These results indicate that the geometry of the cationic peptide component can have a profound influence on the stimulatory capabilities of the TLR2 agonist and subsequently their ability to activate DCs and induce immunity. Susceptibility to proteolytic degradation and serum stability of lipopeptides Another possible factor that could account for the different biological efficacies exhibited by these lipopeptides may be related to their stability under physiological conditions. In light of previous observations that the linear Pam2Cys containing lipopeptide, Pam2CysSK4, is more susceptible to proteolytic degradation than R4Pam2CysB 19, we determined if this was also the case for R4Pam2CysL. Analysis of R4Pam2CysL by RP-HPLC showed that while it elutes as a single major peak at ~38 minutes (peak 1 in Fig 5A), this peak disappears and two smaller peaks (peaks 2 and 3) appear following exposure to trypsin. Analysis of these products by mass spectrometry indicated that they have mass values corresponding to truncated forms lacking three (peak 2) or four (peak 3) R residues. In contrast, while both R4Pam2CysB and R4Pam2CysP present as single major peaks (peak 1 in Fig 5B and peak 1 in Fig 5C, respectively), no breakdown products were detected in the presence of trypsin indicating that these branched lipopeptides are more resistant to degradation. We next examined the stability of these lipopeptides under more physiologically relevant conditions by incubation in the presence of murine serum obtained from naïve animals. By comparing the area of peaks (AUC values) corresponding to each lipopeptide in the LC/MS chromatograms before and after incubation with mouse serum, we detected a significant decrease of R4Pam2CysL but not R4Pam2CysB and R4Pam2CysP (Table 2). Mass spectral analyses also shows the presence of 12


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products corresponding to the loss of 3 and 4 R residues from R4Pam2CysL whereas no evidence of breakdown species relating to R4Pam2CysB and R4Pam2CysP were observed. These results therefore suggest that R4Pam2CysL is more susceptible to degradation in serum and truncation within its cationic antigen binding region could potentially result in its detachment from an antigen. In vivo antigen presentation Given that susceptibility to degradation upon exposure to the physiological environment may follow vaccination and that TLR2 engagement at the surface of DCs precedes activation of T cell responses, we determined whether the differences in susceptibility to proteolytic digestion correlated with the ability of the lipopeptides to facilitate in vivo antigen-presentation to CD8+ T cells. To do this, CFSE-labelled OT-1 cells were adoptively transferred into naïve mice 1 day prior to inoculation with OVA formulated with lipopeptide. Analysis of dividing OT-1 cells, by measuring the progressive loss of CFSE in the draining inguinal lymph nodes of vaccinated animals, was determined 3 days later. Although vaccinations carried out using OVA formulated with either lipopeptide resulted in proliferation of significantly more T cells compared to OVA alone, there was a clear hierarchy of responses with the branched lipopeptides R4Pam2CysB and R4Pam2CysP being more effective than linear R4Pam2CysL (Fig 6A and 6B). Similarly, higher frequencies of dividing and non-dividing OT-I T cells expressing the early activation markers CD44 (Fig 6C) and CD25 (Fig 6D) were detected in animals vaccinated with R4Pam2CysB and R4Pam2CysP. Furthermore, the mean cell expression levels of these markers were higher on these cells than those derived from animals vaccinated with antigen formulated with R4Pam2CysL (Fig 6E).

13



Page 14 of 34

DISCUSSION The use of TLR agonists to facilitate the delivery of vaccine antigens to TLRs expressed on dendritic cells (DCs) has proven to be an effective approach for improving immunogenicity. Our studies as well as those of others have demonstrated that these approaches are more likely to be effective, particularly for the induction of CD8+ T cell responses, if both TLR agonist and antigen are electrostatically or covalently linked

3-7.

In the case of the lipopeptide R4Pam2Cys, electrostatic

association with antigen is facilitated by the peptide component of the molecule which has a branched arrangement of R residues providing positively charged functional groups clustered at the N-terminus. The ability of R4Pam2Cys to bind spontaneously to antigen is an important property precluding the requirement for chemical (covalent) linkage between antigen and adjuvant thereby simplifying vaccine manufacture. The importance of the branched design of R4Pam2CysB and R4Pam2CysP was highlighted in comparative studies with R4Pam2CysL which demonstrated that when R residues are arranged as a contiguous linear sequence as in R4Pam2CysL, the effects of dendritic cell maturation, antigen uptake and induction of pro-inflammatory cytokine secretion are diminished resulting in decreased CD8+ T cell responses. Notwithstanding the fact that all three lipopeptide configurations appear to bind antigen to form similar sized complexes, the manner in which they interact with the molecular surfaces of an antigen may play an influential role in immunogenicity. The guanidinium group of each R residue possesses a planar geometric structure which together with intrinsic chemical and electrical properties facilitate hydrogen bonding and electrostatic (salt-bridge) interactions at five possible sites

27.

In

addition, the three methylene (methanediyl) bridges present within the side chain of each R also provides opportunities to participate in hydrophobic as well as salt bridge interactions. The branched geometries of R4Pam2CysB and R4Pam2CysP, allow the -guanidinium groups and -amino groups of each of the four R residues to be available for electrostatic and hydrogen bond interactions. In the case of R4Pam2CysL, however, only the guanidinium group of each R residue and the single exposed  amino group of the N-terminal R is exposed. The benefits of these branched geometries may also provide flexibility and spacing needed for optimal interaction with molecular surfaces present on antigen compared to more limited movement of the residues when arranged linearly. It has been demonstrated for example that branched peptides with three-lysine K2K cores have asymmetric combinations of short α and long ε primary amino groups which result in branch points that confer flexibility 28. This, together with the possibility that multiple electrostatic interactions can occur between an antigen and the functional groups of each R residue in R4Pam2CysB and R4Pam2CysP due to their available charge, could therefore act together to achieve greater binding stability and increased avidity.

14


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Our results also suggest that R4Pam2CysL is more susceptible to proteolytic hydrolysis upon exposure to trypsin whereas this does not appear to be the case for R4Pam2CysB and R4Pam2CysP. These results are consistent with the work of others that have demonstrated that peptides in branched conformations are not only more stable under physiological conditions but also more resistant to protease activity

28-31.

Trace breakdown products detected following exposure of R4Pam2CysL to

trypsin (or serum [data not shown]) represent forms of the lipopeptide with truncations within its cationic antigen binding region. This suggests the possibility that interactions between R4Pam2CysL and antigen could be disrupted under physiological conditions and that R4Pam2CysB/R4Pam2CysP bound-antigen are more likely to be taken up by cells as a single complex as reflected in their ability to facilitate greater dendritic cell-mediated uptake of antigen than R4Pam2CysL. In light of our results demonstrating that R4Pam2CysB is more effective than R4Pam2CysL at mediating signalling through TLR2, it is also possible that the factors described above could affect the inherent ability of these lipopeptides to engage the TLR2 receptor. Several studies have demonstrated that single amino acid alterations to peptide extensions flanking a TLR2 agonist can significantly affect their recognition by TLR2/6 heterodimers and the ensuing biological activity 32,33. Because our experiments describing TLR2 mediated signalling by the Pam2Cys-based agonists (Fig. 4D) were performed in the absence of antigen and because R4Pam2CysB exhibits a greater electrostatic charge than R4Pam2CysL, the branched version could facilitate increased, non-specific, electrostatic interaction with negatively charged cell surface components (eg. sialic acid containing cell surface glycoproteins). In this context it is relevant to note that peptides containing multiple arginine residues exhibit characteristics including their efficient entry into cells, which could as a consequence enhance the immunogenicity of peptide epitopes

22,34,35

by using entry mechanisms

additional to the receptor-specific transport offered by TLR2. Given the important role that DCs play in priming naïve T cells, it is not surprising that the hierarchy of effects exerted by these lipopeptides on DCs correlate with their ability to facilitate in vivo antigen presentation to CD8+ T cell responses and also improve the magnitude and quality of subsequent CD8+ T cell responses induced. Notable differences between the branched and linear lipopeptide were observed not only in their ability to activate DCs but also facilitate antigen uptake and induce IL-6, TNF- and IL-2 production. These cytokines are known to promote inflammation to alert the immune system of a possible pathogenic threat. IL-4 and IL-10, however, have opposite effects that are anti-inflammatory and more aligned with immunosuppression whilst IL-17 is a major cytokine secreted by a particular subset of T cells and not DCs. In this regard, the increase in IL-6, TNF- and IL-2 but not IL-4, IL-10 and IL-17 induced by these lipopeptides are in line with other reports that Pam2Cys as well as other TLR agonists are potent stimulators of DCs 36

15



Page 16 of 34

We have also found that R4Pam2CysB alone or R4Pam2CysB formulated with antigen do not differ in their ability to activate dendritic cells indicating that any structural changes or effects that may arise from complex formation between lipopeptide and antigen do not appear to influence dendritic cells (data not shown). Even though B cell responses can be mediated by activated DCs that stimulate CD4+ T cells to generate signals or produce cytokines to support B cell differentiation into antibody-producing cells, only marginal differences in antibody responses induced by any of the lipopeptides examined were observed. The apparent independence of antibody induction on lipopeptide geometry may be explained by the fact that direct stimulation of TLRs present on B cells 37-39 plays a contributing role in B cell activation and differentiation into plasma cells. Differences in immunogenicity exhibited by branched and linear peptide epitope-based immunogens have been reported in previous studies conducted by us and others 13,14,40. Of relevance to the present work are findings indicating that branched Pam2Cys-conjugated peptide T- and B-cell determinants are more effective at activating CD4+ and CD8+ T cells in vivo 40. Renaudet et al have also reported advantages conferred by the use of linear but not branched glycolipid-containing peptides; their structures, however, are cyclic and branched 13. Our findings suggest that in addition to the strong adjuvanting properties bestowed particularly by the lipopeptides described here, the inherent stability conferred against in vivo degradation by the branched design could improve vaccine efficacy, dose sparing effects promoting their clinical utility. This is especially relevant given that Pam2Cys is recognised by human TLR2

26

and previously evaluated as a therapeutic vaccine

candidate against chronic hepatitis C virus infection

41-43

highlighting the translational potential of

these findings.

Supporting information. Included as “Supplementary Figure 1.docx”

16


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References (1) (2) (3)

(4)

(5) (6)

(7)

(8)

(9) (10)

(11)

(12) (13)

(14)

Dowling, J. K.; Mansell, A. Toll-Like Receptors: the Swiss Army Knife of Immunity and Vaccine Development. Clin Transl Immunology 2016, 5 (5), e85. Tartey, S.; Takeuchi, O. Pathogen Recognition and Toll-Like Receptor Targeted Therapeutics in Innate Immune Cells. Int. Rev. Immunol. 2017, 36 (2), 57–73. Zom, G. G.; Khan, S.; Britten, C. M.; Sommandas, V.; Camps, M. G. M.; Loof, N. M.; Budden, C. F.; Meeuwenoord, N. J.; Filippov, D. V.; van der Marel, G. A.; Overkleeft, H. S.; Melief, C. J. M.; Ossendorp, F. Efficient Induction of Antitumor Immunity by Synthetic Toll-Like Receptor Ligand-Peptide Conjugates. Cancer Immunol Res 2014, 2 (8), 756–764. Chua, B. Y.; Pejoski, D.; Turner, S. J.; Zeng, W.; Jackson, D. C. Soluble Proteins Induce Strong CD8+ T Cell and Antibody Responses Through Electrostatic Association with Simple Cationic or Anionic Lipopeptides That Target TLR2. J. Immunol. 2011, 187 (4), 1692–1701. Maurer, T.; Heit, A.; Hochrein, H.; Ampenberger, F.; O'Keeffe, M.; Bauer, S.; Lipford, G. B.; Vabulas, R. M.; Wagner, H. CpG-DNA Aided Cross-Presentation of Soluble Antigens by Dendritic Cells. Eur. J. Immunol. 2002, 32 (8), 2356–2364. Aboutorabian, S.; Hakimi, J.; Boudet, F.; Montano, S.; Dookie, A.; Roque, C.; Ausar, S. F.; Rahman, N.; Brookes, R. H. A High Ratio of IC31(®) Adjuvant to Antigen Is Necessary for H4 TB Vaccine Immunomodulation. Hum Vaccin Immunother 2015, 11 (6), 1449– 1455. Prajeeth, C. K.; Jirmo, A. C.; Krishnaswamy, J. K.; Ebensen, T.; Guzman, C. A.; Weiss, S.; Constabel, H.; Schmidt, R. E.; Behrens, G. M. N. The Synthetic TLR2 Agonist BPPcysMPEG Leads to Efficient Cross-Priming Against Co-Administered and Linked Antigens. Eur. J. Immunol. 2010, 40 (5), 1272–1283. Sekiya, T.; Yamagishi, J.; Gray, J. H. V.; Whitney, P. G.; Martinelli, A.; Zeng, W.; Wong, C. Y.; Sugimoto, C.; Jackson, D. C.; Chua, B. Y. PEGylation of a TLR2-Agonist-Based Vaccine Delivery System Improves Antigen Trafficking and the Magnitude of Ensuing Antibody and CD8(+) T Cell Responses. Biomaterials 2017, 137, 61–72. Chua, B. Y.; Olson, M. R.; Bedoui, S.; Sekiya, T.; Wong, C. Y.; Turner, S. J.; Jackson, D. C. The Use of a TLR2 Agonist-Based Adjuvant for Enhancing Effector and Memory CD8 T-Cell Responses. Immunol. Cell Biol. 2014. Chua, B. Y.; Johnson, D.; Tan, A.; Earnest-Silveira, L.; Sekiya, T.; Chin, R.; Torresi, J.; Jackson, D. C. Hepatitis C VLPs Delivered to Dendritic Cells by a TLR2 Targeting Lipopeptide Results in Enhanced Antibody and Cell-Mediated Responses. PLoS ONE 2012, 7 (10), e47492. Chua, B. Y.; Wong, C. Y.; Mifsud, E. J.; Edenborough, K. M.; Sekiya, T.; Tan, A. C. L.; Mercuri, F.; Rockman, S.; Chen, W.; Turner, S. J.; Doherty, P. C.; Kelso, A.; Brown, L. E.; Jackson, D. C. Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-SystemMediated Protection and Subsequent Long-Term Adaptive Immunity. MBio 2015, 6 (6). Mifsud, E. J.; Tan, A. C.; Short, K. R.; Brown, L. E.; Chua, B. Y.; Jackson, D. C. Reducing the Impact of Influenza-Associated Secondary Pneumococcal Infections. Immunol. Cell Biol. 2015. Renaudet, O.; Dasgupta, G.; Bettahi, I.; Shi, A.; Nesburn, A. B.; Dumy, P.; BenMohamed, L. Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation Pathways and Generate Different Magnitudes of Antitumor Immunity. PLoS ONE 2010, 5 (6), e11216. Fitzmaurice, C. J.; Brown, L. E.; McInerney, T. L.; Jackson, D. C. The Assembly and Immunological Properties of Non-Linear Synthetic Immunogens Containing T-Cell and BCell Determinants. Vaccine 1996, 14 (6), 553–560.

17



(15) (16)

(17)

(18)

(19) (20) (21) (22) (23)

(24) (25) (26)

(27) (28) (29)

(30)

Page 18 of 34

Fitzmaurice, C. J.; Brown, L. E.; Kronin, V.; Jackson, D. C. The Geometry of Synthetic Peptide-Based Immunogens Affects the Efficiency of T Cell Stimulation by Professional Antigen-Presenting Cells. International Immunology 2000, 12 (4), 527–535. Cubillos, C.; la Torre, de, B. G.; Jakab, A.; Clementi, G.; Borrás, E.; Bárcena, J.; Andreu, D.; Sobrino, F.; Blanco, E. Enhanced Mucosal Immunoglobulin a Response and Solid Protection Against Foot-and-Mouth Disease Virus Challenge Induced by a Novel Dendrimeric Peptide. J. Virol. 2008, 82 (14), 7223–7230. Cubillos, C.; la Torre, de, B. G.; Bárcena, J.; Andreu, D.; Sobrino, F.; Blanco, E. Inclusion of a Specific T Cell Epitope Increases the Protection Conferred Against Foot-and-Mouth Disease Virus in Pigs by a Linear Peptide Containing an Immunodominant B Cell Site. Virol. J. 2012, 9 (1), 66. Wang, G.-Z.; Tang, X.-D.; Lü, M.-H.; Gao, J.-H.; Liang, G.-P.; Li, N.; Li, C.-Z.; Wu, Y.Y.; Chen, L.; Cao, Y.-L.; Fang, D.-C.; Yang, S.-M. Multiple Antigenic Peptides of Human Heparanase Elicit a Much More Potent Immune Response Against Tumors. Cancer Prev Res (Phila) 2011, 4 (8), 1285–1295. Wijayadikusumah, A. R.; Sullivan, L. C.; Jackson, D. C.; Chua, B. Y. Structure-Function Relationships of Protein-Lipopeptide Complexes and Influence on Immunogenicity. Amino Acids 2017, 26, 6132. Khurshid, S.; Saridakis, E.; Govada, L.; Chayen, N. E. Porous Nucleating Agents for Protein Crystallization. Nat Protoc 2014, 9 (7), 1621–1633. Winzler, C.; Rovere, P.; Rescigno, M.; Granucci, F.; Penna, G.; Adorini, L.; Zimmermann, V. S.; Davoust, J.; Ricciardi-Castagnoli, P. Maturation Stages of Mouse Dendritic Cells in Growth Factor-Dependent Long-Term Cultures. J. Exp. Med. 1997, 185 (2), 317–328. Chua, B. Y.; Eriksson, E. M.; Poole, D. P.; Zeng, W.; Jackson, D. C. Dendritic Cell Acquisition of Epitope Cargo Mediated by Simple Cationic Peptide Structures. Peptides 2008, 29 (6), 881–890. Heinzel, F. P.; Sadick, M. D.; Holaday, B. J.; Coffman, R. L.; Locksley, R. M. Reciprocal Expression of Interferon Gamma or Interleukin 4 During the Resolution or Progression of Murine Leishmaniasis. Evidence for Expansion of Distinct Helper T Cell Subsets. J. Exp. Med. 1989, 169 (1), 59–72. Chen, X.; Oppenheim, J. J.; Howard, O. M. Z. BALB/C Mice Have More CD4+CD25+ T Regulatory Cells and Show Greater Susceptibility to Suppression of Their CD4+CD25Responder T Cells Than C57BL/6 Mice. J. Leukoc. Biol. 2005, 78 (1), 114–121. Mourglia-Ettlin, G.; Merlino, A.; Capurro, R.; Dematteis, S. Susceptibility and Resistance to Echinococcus Granulosus Infection: Associations Between Mouse Strains and Early Peritoneal Immune Responses. Immunobiology 2016, 221 (3), 418–426. Jackson, D. C.; Lau, Y. F.; Le, T.; Suhrbier, A.; Deliyannis, G.; Cheers, C.; Smith, C.; Zeng, W.; Brown, L. E. A Totally Synthetic Vaccine of Generic Structure That Targets Toll-Like Receptor 2 on Dendritic Cells and Promotes Antibody or Cytotoxic T Cell Responses. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (43), 15440–15445. Borders, C. L.; Broadwater, J. A.; Bekeny, P. A.; Salmon, J. E.; Lee, A. S.; Eldridge, A. M.; Pett, V. B. A Structural Role for Arginine in Proteins: Multiple Hydrogen Bonds to Backbone Carbonyl Oxygens. Protein Sci. 1994, 3 (4), 541–548. Tam, J. P.; Lu, Y.-A.; Yang, J.-L. Antimicrobial Dendrimeric Peptides. Eur. J. Biochem. 2002, 269 (3), 923–932. Bracci, L.; Lozzi, L.; Pini, A.; Lelli, B.; Falciani, C.; Niccolai, N.; Bernini, A.; Spreafico, A.; Soldani, P.; Neri, P. A Branched Peptide Mimotope of the Nicotinic Receptor Binding Site Is a Potent Synthetic Antidote Against the Snake Neurotoxin Alpha-Bungarotoxin. Biochemistry 2002, 41 (32), 10194–10199. Dewan, P. C.; Anantharaman, A.; Chauhan, V. S.; Sahal, D. Antimicrobial Action of Prototypic Amphipathic Cationic Decapeptides and Their Branched Dimers. Biochemistry 2009, 48 (24), 5642–5657. 18


Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(31) (32)

(33) (34) (35) (36) (37) (38) (39) (40) (41)

(42) (43)


Liu, S. P.; Zhou, L.; Lakshminarayanan, R.; Beuerman, R. W. Multivalent Antimicrobial Peptides as Therapeutics: Design Principles and Structural Diversities. Int J Pept Res Ther 2010, 16 (3), 199–213. Okusawa, T.; Fujita, M.; Nakamura, J.-I.; Into, T.; Yasuda, M.; Yoshimura, A.; Hara, Y.; Hasebe, A.; Golenbock, D. T.; Morita, M.; Kuroki, Y.; Ogawa, T.; Shibata, K.-I. Relationship Between Structures and Biological Activities of Mycoplasmal Diacylated Lipopeptides and Their Recognition by Toll-Like Receptors 2 and 6. Infect. Immun. 2004, 72 (3), 1657–1665. Takeda, Y.; Azuma, M.; Hatsugai, R.; Fujimoto, Y.; Hashimoto, M.; Fukase, K.; Matsumoto, M.; Seya, T. The Second and Third Amino Acids of Pam2 Lipopeptides Are Key for the Proliferation of Cytotoxic T Cells. Innate Immun 2018, 24 (5), 323–331. Buschle, M.; Schmidt, W.; Zauner, W.; Mechtler, K.; Trska, B.; Kirlappos, H.; Birnstiel, M. L. Transloading of Tumor Antigen-Derived Peptides Into Antigen-Presenting Cells. Proceedings of the National Academy of Sciences 1997, 94 (7), 3256–3261. Lührs, P.; Schmidt, W.; Kutil, R.; Buschle, M.; Wagner, S. N.; Stingl, G.; Schneeberger, A. Induction of Specific Immune Responses by Polycation-Based Vaccines. The Journal of Immunology 2002, 169 (9), 5217–5226. Mifsud, E. J.; Tan, A. C. L.; Jackson, D. C. TLR Agonists as Modulators of the Innate Immune Response and Their Potential as Agents Against Infectious Disease. Front Immunol 2014, 5, 79. Glaum, M. C.; Narula, S.; Song, D.; Zheng, Y.; Anderson, A. L.; Pletcher, C. H.; Levinson, A. I. Toll-Like Receptor 7-Induced Naive Human B-Cell Differentiation and Immunoglobulin Production. J. Allergy Clin. Immunol. 2009, 123 (1), 224–230.e224. Richard, K.; Pierce, S. K.; Song, W. The Agonists of TLR4 and 9 Are Sufficient to Activate Memory B Cells to Differentiate Into Plasma Cells in Vitro but Not in Vivo. J. Immunol. 2008, 181 (3), 1746–1752. Fillatreau, S.; Manz, R. A. Tolls for B Cells. Eur. J. Immunol. 2006, 36 (4), 798–801. Fitzmaurice, C. J.; Brown, L. E.; Kronin, V.; Jackson, D. C. The Geometry of Synthetic Peptide-Based Immunogens Affects the Efficiency of T Cell Stimulation by Professional Antigen-Presenting Cells. International Immunology 2000, 12 (4), 527–535. Chua, B. Y.; Healy, A.; Cameron, P. U.; Stock, O.; Rizkalla, M.; Zeng, W.; Torresi, J.; Brown, L. E.; Fowler, N. L.; Gowans, E. J.; Jackson, D. C. Maturation of Dendritic Cells with Lipopeptides That Represent Vaccine Candidates for Hepatitis C Virus. Immunol. Cell Biol. 2003, 81 (1), 67–72. Chua, B. Y.; Eriksson, E. M.; Brown, L. E.; Zeng, W.; Gowans, E. J.; Torresi, J.; Jackson, D. C. A Self-Adjuvanting Lipopeptide-Based Vaccine Candidate for the Treatment of Hepatitis C Virus Infection. Vaccine 2008, 26 (37), 4866–4875. Gowans, E. J.; Roberts, S.; Jones, K.; Dinatale, I.; Latour, P. A.; Chua, B.; Eriksson, E. M. Y.; Chin, R.; Li, S.; Wall, D. M.; Sparrow, R. L.; Moloney, J.; Loudovaris, M.; Ffrench, R.; Prince, H. M.; Hart, D.; Zeng, W.; Torresi, J.; Brown, L. E.; Jackson, D. C. A Phase I Clinical Trial of Dendritic Cell Immunotherapy in HCV-Infected Individuals. Journal of Hepatology 2010, 53 (4), 599–607.

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Page 20 of 34

Table 1. Analysis of formulations using dynamic light scattering* Sample

Hydrodynamic radius

% Pda^

(nm ±SD) OVA

18.5 ±2.4

>20%

R4Pam2CysB

32.9 ±4.7

>20%

R4Pam2CysP

46.3 ±3.8

>20%

R4Pam2CysL

21.1 ±3.3

>20%

OVA+R4Pam2CysB

596.9 ±44.4

18.8%

OVA+R4Pam2CysP

502.7 ±72.9

18.6%

OVA+R4Pam2CysL

478.0 ±69.4

17.8%

*Results

were derived from measurements from duplicate samples from 2

separate experiments and each result represents the mean of 30 measurements performed at 25°C. ^Pda = polydispersity. A peak with < 20% polydispersity is considered to be monodispersed.

20


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Table 2. Compound stability in mouse serum Sample

% reduction of AUC following

Breakdown products

exposure to serum*

detected (Da)#

R4Pam2CysB

2.2% ±1.1

ND

R4Pam2CysP

3.3% ±1.7

ND

R4Pam2CysL

63.1% ±4.7

1,129.8 (corresponding to loss of 3 R) 974.3 (corresponding to loss of 4 R)

* Results

are derived from area under the curve (AUC) measurements of each compound in LC-MS

chromatograms obtained before and after incubation with mouse serum for 18 hours at 37°C followed by precipitation with tricholoracetic acid 15. #Species

corresponding to degradation of each compound through loss of arginine residues (R).

ND=not detected. Da=Daltons

21



Page 22 of 34

Figure 1. Diagrammatic representation of the branched R4Pam2Cys variants and their association with OVA to form complexes. (A) The branched lipopeptide R4Pam2Cys (R4Pam2CysB) was synthesized using a scaffold of lysine (K) residues to which four arginine (R) residues are attached to the α and ε-amino groups of each Nterminal K. Variants of this design include; (B) a lipopeptide incorporating 4 R residues with each attached to the ε-amino group of K residues arranged in a contiguous sequence (R4Pam2CysP) or (C) where the 4 R residues are arranged contiguously (R4Pam2CysL). Each lipopeptide contains the TLR2 agonist Pam2Cys attached through 2 serine residues (S2) to the ε-amino group of the C-terminal K. (D-F) To analyse the electrostatic association of these lipopeptides with antigen, 10µg of OVA alone, OVA mixed with lipopeptide at a 3-fold molar excess or the same amount of each lipopeptide alone, were formulated separately in 50µl saline. The size distribution of particles formed within these solutions was measured by dynamic light scattering. 22


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Figure 2. Immune responses induced by OVA formulated with R4Pam2CysP or R4Pam2CysB and their effects on dendritic cells (A) For measuring antibody responses, BALB/c mice (n=5/group) were vaccinated s.c. with 25 g of OVA alone or OVA formulated with a 3-fold molar excess of lipopeptide on day 0 and again on day 21. Serum OVA-specific antibody titres (±SD) 14 days after the second dose were determined by ELISA. (B) To measure CD8+ T cell responses, C57BL/6 mice (n=3/group) were vaccinated in a similar manner. Spleens were removed 7 days later and IFN-γ secretion from OVA257-264-specific CD8+ T cells enumerated by intracellular cytokine staining. (C) To measure in vivo cytolytic responses, CFSE-labelled splenocytes composed of OVA257-264-pulsed CFSEhigh and irrelevant peptide-pulsed CFSElow cells were injected intravenously 7 days following vaccination. Spleens were harvested 16 hours later and the percentage target cell lysis (±SD) measured by determining frequencies of OVA257-264-pulsed cells in relation to irrelevant peptide-pulsed cells. (D) D1 cells (2×105) were incubated with OVA alone or OVA formulated with a 3-fold molar excess of each lipopeptide. Cells were also incubated with LPS (5g/mL) or used untreated as controls. MHC class IIhigh activated cells were measured 16 hours later and results presented as the mean percentage (%±SD) from each culture (n=3/group). (E) To examine antigen uptake, cells were incubated with 5g of FITC-conjugated OVA alone (OVA-FITC) or OVA formulated with lipopeptide (n=3/group). Following quenching of extracellular fluorescence, the number of cells (±SD) harbouring intracellular fluorescence were counted 16 hours later. Asterisks (*) indicate p values of

The geometry of a TLR2-agonist-based adjuvant can affect the

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