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In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity

Nature Biotechnology volume 33, pages 1201–1210 (2015)Cite this article

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Abstract

The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer–TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer–TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer–TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer–TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.

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Figure 1: Increasing densities of TLR-7/8a arrayed on polymer carriers is associated with particle formation and enhanced lymph node cytokine production.
Figure 2: Particle formation by Poly-7/8a enhances local retention and promotes uptake by migratory APCs.
Figure 3: Particle-forming Poly-7/8a induce high-magnitude and persistent local innate immune activation that is associated with enhanced CD8+ T cell responses and TH1-skewed antibody responses.
Figure 4: Persistent, local innate immune activation is necessary and sufficient for eliciting protective CD8+ and TH1 CD4+ T cell responses.
Figure 5: Temperature-responsive polymer particles (TRPP) permit temperature-dependent particle assembly that leads to persistent innate immune activation and protective CD8+ T cell responses.
Figure 6: Co-delivery of TLR-7/8a and protein antigen on a self-assembling temperature-responsive vaccine particle.

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Acknowledgements

The authors wish to acknowledge M. Dillon, K. Wuddie and C. Chiedi at the Vaccine Research Center and B. Klaunberg and V. Diaz at the Mouse Imaging Facility (MIF) for their valuable support and assistance with the animal studies. We would also like to thank K. Ulbrich, R. Swenson and G. Griffiths for their support and helpful insights. This work was supported in part by the BIOPOL project (Grant of the Ministry of Education, Youth and Sports of the Czech Republic, no. EE2.3.30.0029); by the Czech Science Foundation (15-15181S); by Charles University (UNCE 204025/2012); by a Cancer Research UK grant (C552/A17720); and by the Office of AIDS Research and the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health.

Author information

Author notes

  1. Geoffrey M Lynn and Richard Laga: These authors contributed equally to this work.

  2. Leonard W Seymour and Robert A Seder: These authors jointly supervised this work.

Authors and Affiliations

  1. Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA

    Geoffrey M Lynn, Patricia A Darrah, Andrew S Ishizuka, Alexandra J Balaci, Ayako Yamamoto, Connor R Buechler, Kylie M Quinn, Kathrin Kastenmüller, Joseph R Francica & Robert A Seder

  2. Department of Oncology, University of Oxford, Oxford, UK

    Geoffrey M Lynn, Richard Laga, Ryan Cawood, Thomas Hills, Kerry D Fisher & Leonard W Seymour

  3. Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Richard Laga, Michal Pechar, Robert Pola & Tomas Etrych

  4. Imaging Probe Development Center, National Heart, Lung, and Blood Institute,

    Andrés E Dulcey & Olga Vasalatiy

  5. NIH, Rockville, Maryland, USA

    Andrés E Dulcey & Olga Vasalatiy

  6. Lymphocyte Biology Section, Laboratory of Systems Biology, NIAID, NIH, Bethesda, Maryland, USA

    Michael Y Gerner

  7. Biological Imaging Section, Research Technologies Branch, NIAID, NIH, Bethesda, Maryland, USA

    Margery G Smelkinson

  8. Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic

    Ondrej Vanek

  9. Department of Chemistry, University of California, Irvine, Irvine, California, USA.,

    Lalisa Stutts, Janine K Tom, Keun Ah Ryu & Aaron P Esser-Kahn

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  1. Geoffrey M Lynn
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Contributions

G.M.L., R.L., K.D.F., L.W.S. and R.A.S were involved in experimental planning, interpreting data and writing the manuscript. G.M.L., R.L., A.E.D., O. Vasalatiy, J.K.T., L.S., K.A.R. and A.P.E.-K. planned and carried out the synthesis, purification and characterization of small molecules. R.L., R.P., M.P., T.E., O. Vanek and G.M.L. planned and completed the synthesis, purification and characterization of the polymer precursors and polymer conjugates. P.A.D, A.S.I., A.J.B., A.Y., K.M.Q., C.R.B., K.K. and J.R.F. planned and conducted many of the biological studies. M.Y.G. and M.G.S. carried out the confocal microscopy studies on lymph node sections and polymer particles, respectively. T.H. and R.C. developed the plasmids to express the HIV Gag-coil fusion protein. R.L., M.P., R.P. and T.E. devised the coil-coil strategy. A.P.E.-K., T.E., K.D.F., L.W.S. and R.A.S. are principal investigators who advised the studies.

Corresponding author

Correspondence to Robert A Seder.

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Competing interests

G.M.L., R.L., K.D.F., L.W.S. and R.A.S. are listed as inventors on patents describing polymer-based vaccines. K.D.F. and L.W.S. are scientific founders and equity holders in PsiOxus Therapeutics, Ltd. (Oxford, UK). G.M.L. and J.R.F. are scientific founders and equity holders in Avidea Technologies, Inc., which is developing polymer-based technologies for immunotherapeutic applications (Baltimore, Maryland, USA).

Integrated supplementary information

Supplementary Figure 1 Synthesis of polymer-TLR-7/8a conjugates (Poly-7/8a).

(a) Chemical structures of imidazoquinoline-based TLR-7/8a. Polymer reactive analogs of the commercially available TLR-7/8a, R848, were produced by replacing the isopropanol group with reactive linkers that are indicated by the shaded boxes overlaying the structures of SM 7/8a, SM 7/8a-alkane and SM 7/8a-PEG. Note that the alkane and PEG linkers are of comparable length but different composition (hydrophobic vs. hydrophilic). The terminal amine on each of the linkers permitted facile attachment to amine reactive polymer precursors. (b) Poly-7/8a were generated by reacting nucleophilic TLR-7/8a (e.g., SM 7/8a) with HPMA-based copolymers in a one step reaction, resulting in a stable amide bond between the TLR-7/8a and the polymer backbone. Note that the brackets represent repeating units of each monomer, with the subscripts, x and y, representing the percentage composition (mol%) of each monomer. Poly = polymer; SM = small molecule; HPMA = N-(2-hydroxypropyl)methacrylamide; MA = methacrylamide; Ahx = aminohexanoic acid; PEG = Polyethylene glycol; TT = 2-Thiazolidine-2-thione.

Supplementary Figure 2 Combinatorial synthesis of Poly-7/8a.

(a) Structures of Imidazoquinoline-based TLR-7/8a used in the generation of combinatorial libraries of Poly-7/8a. In addition to SM 7/8a described previously, a ~ 20-fold more potent TLR-7/8a with a xylene linker was prepared and is referred to as SM 20x7/8a. The potency of the two TLR-7/8a were determined in vitro using HEK293 hTLR7 reporter cells. Absorbance at 620 nm in this experiment is proportional to TLR-7 activity. Note that acetylated versions of the TLR-7/8a were used in these in vitro assays as this best represents the physicochemical characteristics of the compounds when they are attached to the polymers. (b) A combinatorial library of Poly-7/8a was generated by attaching 2 unique TLR-7/8a (SM 7/8a or SM 20x7/8a) to reactive HPMA-based copolymers at different densities (~ 2, 4, 8 mol %) using short, alkane or PEG linkers. By reacting 2 unique TLR-7/8a at 3 different densities with 3 different linkers, 18 unique products can be generated, as illustrated (c). Note that this cartoon representation is for illustrative purposes; not all Poly-7/8a represented in this schematic were evaluated in this study, nor does this schematic represent all the materials described herein.

Supplementary Figure 3 Screening a combinatorial library of Poly-7/8a in vivo.

Combinatorial library of Poly-7/8a with varying TLR-7/8a density and linker group composition. (b) Cartoon schematic of a combinatorial library of Poly-7/8a. (c) Poly-7/8a normalized for TLR-7/8a dose (12.5 nmol) were subcutaneously administered into both hind footpads of C57BL/6 mice. After 24 h, draining lymph nodes (n = 4) were harvested and processed to generate a cell suspension that was cultured for 8 h and then evaluated for IP-10 production by ELISA. Data are reported as mean; statistical significance is reported relative to naïve (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 4 Increasing agonist density is associated with particle formation and TLR-7 dependent lymph node cytokine production.

(a) Properties of Poly-7/8a and controls. (b) Negative control polymers were generated using 2-aminopyridine (AP) to account for the contribution of the aromatic amine present on the Imidazoquinoline-based TLR-7/8a. AP was attached to polymers using a PEG or amphiphilic (AMPH) spacer to generate polymer coils and polymer particles, respectively. (c, d) Adjuvants were administered subcutaneously and after 24 h lymph nodes draining the site of immunization were harvested to create cell suspensions that were cultured for 8 h and then evaluated for (c) IFNα or (d) IFNγ by ELISA. (e, f) PP-7/8a (PEG, 10 mol% 7/8a) was administered subcutaneously to wild type (WT) or knockout mice and cytokines were evaluated from lymph node cell suspensions. All data are reported as mean ± SEM; except where indicated, statistical significance is relative to all other groups (ANOVA with Bonferroni correction, n = 4); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01

Supplementary Figure 5 In vivo tracking of dye-labeled Poly-7/8a.

(a) Properties of fluorescent dye-labeled materials. (b) Example gating tree. (c) Gates designating adjuvant positive cells (AF488+) were set relative to naïve. (d) Percent adjuvant uptake by the major CD11c+ DC subsets. (e-g) Evaluation of polymer controls and polymer particles with different densities of TLR-7/8a (3 and 10 mol% 7/8a) reveals that pharmacokinetics and uptake by APCs is primarily dependent on the morphology of the carrier (i.e. submicron particle) and is independent of the attached agonist. All data are reported as mean ± SEM. DC = dendritic cell; pDC = plasmacytoid dendritic cell; Mac = Macrophage; Mon = monocyte.

Supplementary Figure 6 Characterization of DC populations in draining lymph nodes and spleen.

AF488-labeled materials normalized for dose of TLR-7/8a (62.5 nmol) were unilaterally administered subcutaneously into the hind footpad of C57BL/6 mice. (a-c) At serial timepoints thereafter, lymph nodes (n = 3) or spleen (n = 1) were isolated and enzyme-digested to create cell suspensions that were stained and evaluated by flow cytometry. (a) Magnitude of DCs and (b) expression of the costimulatory molecule CD80 were evaluated by flow cytometry. (c) DC populations in the spleen were evaluated for costimulatory molecule expression (CD80 MFI) at serial timepoints. Note that the small molecule TLR-7/8a (SM 7/8a) leads to transient activation of the major DC subsets and B cells in both spleen and lymph nodes, whereas PP-7/8a leads to persistent activation of CD8- B220- DC (monocyte-derived DCs, skin-derived DCs and CD8- resident DCs) and CD8+ DC. Serum (n = 3) was evaluated for the presence of (h) IL-12p40 at serial timepoints. All data are reported as mean ± SEM; except where indicated, significance is relative to all other groups (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 7 Orientation of the TLR-7/8a attached to the polymer carrier influences the timing of onset and magnitude of lymph node cytokine production.

Poly-7/8a were prepared with TLR-7/8a attached to the polymer carrier with either the C4-amine exposed (1) (PP-20x7/8a) or blocked (2) (PP-R20x7/8a). (b, c) Poly-7/8a with two different orientations of TLR-7/8a were administered subcutaneously into the hind footpads of C57BL/6 mice and lymph nodes (n = 4) were isolated at serial timepoints thereafter and cultured overnight. Supernatant from the ex vivo lymph node cell suspensions (n = 4) were evaluated for (b) IL-12 and (c) IP-10 by ELISA. Note that blocking the C4-amine delays onset and leads to lower magnitude of cytokine production. All data are reported as mean ± SEM; significance is relative to all other groups (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 8 Anti-OVA antibody responses.

(a-d) Poly-7/8a, SM 7/8a or a control were formulated with 50 μg of OVA in PBS and given subcutaneously to C57BL/6 mice (n = 5) at days 0 and 14. Serum was collected from vaccinated mice at day 28 and evaluated for anti-OVA IgG1 and IgG2c antibodies. Doses of adjuvant and polymer are provided in the accompanying tables.

Supplementary Figure 9 Particle-forming Poly-7/8a induces locally restricted Th1-polarizing cytokines.

(a, b) R848 (62.5 nmol), PP-7/8a (62.5 nmol) or CpG ODN 1826 (3.1 nmol) were administered subcutaneously into the footpad of mice. Cytokine bead array was used to quantify cytokines present (a) in the serum (n = 5) at 6 h (peak), or (b) from draining lymph nodes (n = 4) at 24 h. All data are reported as mean ± SEM; except where indicated, statistical significance is relative to both OVA alone and OVA + R848 (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 10 Polymer conjugates of TLR-2/6 and TLR-4 agonists.

Structures of polymer conjugates of TLR-2/6 (Pam2Cys) and TLR-4 (Pyrimidoindole) agonists are shown above. Both agonists were linked to the polymer backbones through hydrophilic PEG spacers at > 5 mol % agonist density to induce polymer particle (PP) formation in aqueous conditions. Synthesis and characterization of PP-Pam2Cys and PP-PI conjugates is provided in the Supplementary Materials and Methods.

Supplementary Figure 11 Local and systemic innate immune activation and morbidity by particulate and unconjugated TLRa.

(a-d) TLR-2/6 agonists (PP-Pam2Cys and unconjugated Pam2Cys, 20 nmol), heterocyclic TLR-4 agonists (PP-PI and PI, 20 nmol), lipid-based TLR-4 agonists (50 μg Alum / MPL 5 μg or MPL alone (5 μg, ~3 nmol)), TLR-7/8a (PP-7/8a and R848, 12.5 nmol), TLR-9a (CpG/polyplex and CpG alone, 3 nmol), and controls were delivered subcutaneously into both hind footpads of C57BL/6 mice. Draining lymph nodes were harvested at day 4 (early peak for local activity) and were evaluated for (a) total CD11c+ DCs per lymph node (n = 3) and (b) IL-12p40 production (n = 8). (c) Serum (n = 5) was collected at 4 h (peak for systemic activity) post-immunization and was evaluated for IL-12p40 by ELISA. (d) Percent body weight change (n = 5) at peak (24 h) following subcutaneous administration of different vaccine adjuvants. (e) Meta-analysis of 4 independent studies (n = 43 groups) showing the relationship between systemic IL-12 production and body weight change (relative to time = 0) for mice immunized with either particle carriers of TLRa, unconjugated (free) TLRa or controls. PP = polymer particle; PI = pyrimidoindole; MPL = Monophosphoryl Lipid A; Alum = Aluminum hydroxide; CpG/Polyplex = Poly(Lysine).HCl complexed to CpG ODN 1826 at 20:1 N:P. See Supplementary Materials and Methods for chemical synthesis and formulation of the different TLRa. All data are representative of two or more independent experiments that included multiple time points. Data on linear scale are reported as mean ± SEM and data on log scale are reported as geometric mean ± 95% CI; statistical significance is shown for specific comparisons and for adjuvant formulations relative to naïve and particle controls (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 12 Particle-forming Poly-7/8a elicit protective CD8 T cell responses.

(a-c) Poly-7/8a, SM 7/8a and polymer controls admixed with 50 μg of OVA in 50 μL of PBS were administered subcutaneously into the hind footpad of C57BL/6 mice (n = 6) at days 0 and 14. Three different small molecule TLR-7/8a were evaluated in this experiment: either commercially available R848, SM 7/8a, or SM 20x7/8a. Poly-7/8a were evaluated for either dose, comparing PP-7/8a at 12.5 and 62.5 nmol, or potency of the agonist attached, comparing PP-7/8a with PP-20x7/8a. (b) The proportion of tetramer+ positive CD8 T responses was evaluated from whole blood at day 24. (c) Mice (n = 6) were challenged intravenously at day 28 with Listeria monocytogenes expressing full-length OVA Albumin (LM-OVA) and bacterial burdens in spleen was evaluated at day 31. Note that protection (inverse of bacterial burden) is proportional to the tetramer+ response. All data are reported as mean ± SEM; except where indicated, statistical significance is relative to both OVA alone and OVA + R848 (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 13 Particle forming Poly-7/8a elicit Th1 cells that mediate protection against Leishmania major.

(a-f) C57BL/6 mice received subcutaneous immunizations of 20 μg of MML protein from L major either alone or admixed with an adjuvant on days 0, 21 and 42. (a) Splenocytes were isolated on either (b) day 56 (n = 4) or (c, d) day 70 (n = 5) and stimulated in vitro with a peptide pool derived from MML. Antigen-specific CD4 T cells were evaluated for their capacity to produce Th1 characteristic cytokines (IFNγ, IL-2 or TNFα); (b) and (c) report total cytokine producing CD4 T cells (magnitude), whereas (d) reports the frequency of CD4 T cells producing combinations of IFNγ, IL-2 and TNFα (quality). (e) Mice (n = 6) were challenged intradermally in both ears with L major at day 70. Ear lesion diameters were measured up to 12 weeks after challenge. All data are reported as mean ± SEM; except where indicated, statistical significance is relative to naïve, MML alone and SM 7/8a (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 14 Temperature-responsive particle-forming Poly-7/8a (TRPP-7/8a) induce protective CD8 T cell responses.

(a) First-generation TRPP-7/8a are N-Isopropylacrylamide (NIPAM)-based copolymers. Note that the TLR-7/8a (7/8a or 20x7/8a) or a control ligand (AP) were attached to the NIPAM-based copolymers using a similar reaction scheme as described in supplementary figure 1 (see materials and methods). (b) A series of TRPP-7/8a were produced with increasing densities of either SM 7/8a, SM 20x7/8a or the control, AP-AMPH. Note that increasing densities of the hydrophobic ligands attached to the polymers leads to decreasing transition temperatures, the temperature at which particle formation occurs in aqueous solution. (c, d) TRPP-7/8a and controls were evaluated in a vaccination and challenge model using OVA. C57BL/6 mice (n = 5) received 50 μg of OVA either alone or admixed with adjuvant that was administered subcutaneously in 50 μL of PBS at days 0 and 14. (c) At day 24, the proportion of tetramer+ CD8 T cells was evaluated from whole blood. (d) The capacity of the tetramer+ CD8 T cells to mediate protection was assessed by challenging the mice intravenously at day 28 with LM-OVA. Bacterial burdens were assessed in the spleen at day 31.(e, f, g) Serum was collected from vaccinated mice at day 28 and evaluated for (e) anti-OVA IgG1 and (f) IgG2c antibodies as well as (g) the ratio of the two isotype titers (geometric mean). Data are reported as mean ± SEM; statistical significance is relative to OVA alone (ANOVA with Bonferroni correction); ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Supplementary Figure 15 Self-assembling temperature-responsive vaccine particle.

(a) Schematic of a second-generation di-block copolymer-based TRPP-7/8a used for attachment of TLR-7/8a and a coil peptide. The hydrophilic block consists of HPMA and propargyl(methacrylamide) (PgMA). The acetylene group on PgMA allowed for the attachment of ligands that are modified with azide groups. The hydrophobic block is comprised of poly(diethylene glycol(methacrylate)) homopolymer that allows for the transition temperature to be independent of the attached ligands and contains a biodegradable ester group. Ligands (TLR-7/8a, peptide or fluorophore) were attached to the diblock copolymer through copper-catalyzed 1,3-dipolar cycloaddition. (b) Summary of TRPP-7/8a and TRPP controls. (c) Amino acid sequence of the HIV Gag p41 coil fusion protein; note that the coil domain (KSK) on the protein is complementary to the ESE coil peptide attached to the polymers. (d) Schematic representation of the anti-parallel coil-coil interactions that occur between the ESE and KSK coil peptides.

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Lynn, G., Laga, R., Darrah, P. et al. In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity. Nat Biotechnol 33, 1201–1210 (2015). https://doi.org/10.1038/nbt.3371

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