Structure-based programming of lymph-node targeting in molecular vaccines

Journal name:
Nature
Volume:
507,
Pages:
519–522
Date published:
DOI:
doi:10.1038/nature12978
Received
Accepted
Published online

In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs, where they are efficiently filtered by resident phagocytes1, 2. Here we translate this ‘albumin hitchhiking’ approach to molecular vaccines, through the synthesis of amphiphiles (amph-vaccines) comprising an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Administration of structurally optimized CpG-DNA/peptide amph-vaccines in mice resulted in marked increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumour efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly applicable strategy to simultaneously increase the potency and safety of subunit vaccines.

At a glance

Figures

  1. Design of an LN-targeted molecular adjuvant.
    Figure 1: Design of an LN-targeted molecular adjuvant.

    a, Structure of amph-CpGs. b, SEC of FAM-CpGs alone or after incubation with FBS for 2h (left), and percentage CpG co-migrating with albumin peaks (right). Vertical dashed line provides a guide to the eye. cg, IVIS fluorescence imaging of excised draining LNs from C57BL/6 mice (n = 4 LNs per group) injected with FAM-CpGs (3.3nmol) in soluble form, emulsified in IFA, entrapped in liposomes, or as amphiphile conjugates. c, IVIS images and quantification from inguinal and axillary nodes at 24h. d, CpG accumulation in draining LNs. e, IVIS quantification of CpG in LNs 24h after injection of G-quadruplex-forming Lipo-Gn-CpGs. f, Immunohistochemistry of inguinal LNs 24h after injection (CD3, blue; B220, pink; CpG, green). g, LN CpG+ cells determined by flow cytometry at 24h. ***P<0.001, **P<0.01, *P<0.05 compared with soluble CpG by one-way analysis of variance (ANOVA) with Bonferroni post-test. Data represent mean±standard error of the mean (s.e.m.) of 2–3 independent experiments. NS, not significant.

  2. LN targeting enhances potency while reducing systemic toxicity of CpG.
    Figure 2: LN targeting enhances potency while reducing systemic toxicity of CpG.

    ac, C57BL/6 mice (n = 4–8 per group) were immunized with OVA (10µg) plus CpG (1.24nmol) on day 0 and day 14; shown are SIINFEKL tetramer (a) and intracellular cytokine staining (b) on peripheral blood at day 20. c, LN CpG fluorescence correlation with T-cell response. d, Serum cytokines after injection (n = 3 per group) of 6.2nmol CpG. e, Splenomegaly (n = 3 per group) assessed on day 6 after three injections of CpG. bw, body weight. Scale bar, 1cm. ***P<0.001, **P<0.01, *P<0.05 by one-way ANOVA with Bonferroni post-test. Data show mean±s.e.m. of 2–4 independent experiments. NS, not significant.

  3. Design of LN-targeted amph-peptides.
    Figure 3: Design of LN-targeted amph-peptides.

    a, Structure of amph-peptides. b, Amph-PEG-fluorescein insertion into cell membranes quantified by fluorescence spectroscopy after 1h incubation with splenocytes in the presence of 100µM albumin. c, d, C57BL/6 mice (n = 4 LNs per group) were injected with fluorescent amph-PEGs with varying PEG lengths (fixed C18 diacyl lipid tails; c) or lipid tail lengths (fixed PEG length 48 EG units; d); draining LNs were excised and imaged by IVIS after 24h. ***P<0.001, **P<0.01, *P<0.05 by one-way ANOVA with Bonferroni post-test. Data represent mean±s.e.m. of two independent experiments. NS, not significant.

  4. Amph-vaccines maximize the immunogenicity and therapeutic efficacy of polypeptide vaccines.
    Figure 4: Amph-vaccines maximize the immunogenicity and therapeutic efficacy of polypeptide vaccines.

    ac, C57BL/6 mice (n = 3–4 per group) were immunized with SIV Gag, Trp2, or E7 peptides (10µg) plus CpG (1.24nmol) on day 0 and day 14; shown are tetramer-positive CD8+ T-cells (a) and intracellular cytokine production (b) in peripheral blood on day 20. c, Trp2-specific cytotoxicity measured using an in vivo killing assay on day 21. d, e, Tumour growth in C57BL/6 mice (n = 8 per group) inoculated with 3×105 TC-1 (d) or B16F10 (e) tumour cells and vaccinated with CpG plus E7 peptide or Trp2 peptide (10µg prime, 20µg boost), respectively, on days indicated by arrows. Statistically significant differences between soluble and amph-vaccines are indicated by asterisks: ***P<0.001, **P<0.01, *P<0.05 by one-way ANOVA with Bonferroni post-test. Data show mean±s.e.m. of 2–4 independent experiments. NS, not significant.

  5. Interaction between albumin and amph-CpGs.
    Extended Data Fig. 1: Interaction between albumin and amph-CpGs.

    a, SEC of FBS, albumin and fluorescein-labelled amph-CpGs. FBS and bovine serum albumin (BSA) were monitored using absorptions at 280nm, whereas CpG oligonucleotides were monitored at 480nm (fluorescein peak). b, Lipo-CpG, but not CpG, interacts with serum albumin as shown by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) after protein pull-down assays: FBS was incubated with 3′-biotin-labelled CpG (CpG-biotin), lipo-CpG (lipo-CpG-biotin) or PBS for 1h at 37°C. Streptavidin-conjugated magnetic beads were added to capture biotinylated CpGs and any associated proteins, separated by a magnet, boiled to release bound CpG/proteins, and subjected to SDS–PAGE analysis. Lane 1: protein MW ladder; lane 2: purified BSA; lane 3: FBS (100× dilution, 10μl loading); lane 4: pull-down control, FBS incubated with streptavidin-magnetic beads; lane 5: pull-down with CpG-biotin, FBS was incubated with CpG-biotin and streptavidin magnetic beads; lane 6: pull-down with lipo-CpG-biotin, FBS incubated with lipo-CpG-biotin and streptavidin magnetic beads; lane 7: FBS (100× dilution, 5μl loading). c, Fluorescein-labelled CpG or lipo-CpG was incubated with albumin-conjugated agarose resin for 1h at 37°C, and the resin was separated by filtration. The filtrate and recovered agarose were visualized by a gel imager and quantified by fluorescence measurements. d, Bio-layer interferometry measurements of lipo-CpG and CpG binding to immobilized BSA. Albumin-conjugated BLI probes were immersed in solutions of lipo-CpG, and wavelength shifts (Δλ) of the interferometry pattern association and dissociation curves were followed over time to determine affinity constants of binding at 25°C. Shown are apparent kon, koff and KD values from fits to the data. e, FRET between FAM-labelled lipo-CpG and rhodamine-conjugated albumin (BSA-Rh) assessed by fluorescence spectroscopy. CpG-F or lipo-CpG-F (1.65µM) alone or mixed with BSA-Rh (1.5µM) in PBS were excited at 488nm and emission was recorded from 500–650nm.

  6. Construction and characterization of G-quadruplex-stabilized CpG adjuvants.
    Extended Data Fig. 2: Construction and characterization of G-quadruplex-stabilized CpG adjuvants.

    a, G-quadruplex-stabilized CpG micelles are self-assembled from amphiphiles composed of three distinct segments: an immunostimulatory CpG sequence, a central repeat block containing n = 1–10 G-quartet-forming guanines followed by (10n) non-interacting thymidines, and a diacyl lipid tail. In aqueous solutions, these amphiphiles self-assembled into three-dimensional spherical micelles with a CpG corona and a lipid core. In the presence of K+, neighbouring guanine repeats in the oligonucleotide corona form G-quadruplex structures via Hoogsteen hydrogen bonds and stabilize the micelle structure. The stability of the oligonucleotide micelles in the presence of serum was programmed by altering the length of the guanine repeat. b, Parallel G-quartet formation among DNA strands within the micelles was detected by circular dichroism (CD) spectroscopy, as manifested by the shifting of positive peaks from 278nm towards 262nm and troughs at 245nm as the number of guanines in the structure increased. c, Pyrene excimer fluorescence was used to assay the stabilities of G-quadruplex micelles in the presence of albumin: pyrene dye incorporated in stabilized CpG micelles (n>2) retained excimer fluorescence in the presence of high concentrations of albumin. By contrast, albumin binds to the lipid moiety of unstabilized micelles (n2) and disrupts the micelle structures, leading to loss of excimer fluorescence in an albumin-concentration-dependent manner as the protein disrupts the micelles into albumin-bound unimers. Shown below the schematic is the fraction of amph-CpG remaining in the micellar state as a function of albumin concentration as reported by excimer fluorescence. Arrow indicates the plasma concentration of albumin. d, Stability profiles of G-quadruplex CpG micelles as measured by SEC in the presence of FBS. Fluorescein-labelled CpG micelles were incubated with 20% FBS in PBS in the presence of 10mM Mg2+ and 20mM K+ at 37°C for 2h, then analysed by SEC. FBS and BSA were monitored using absorptions at 280nm, whereas lipo-Gn-CpG amphiphiles were monitored at 480nm (fluorescein peak). Lipo-Gn-CpG with n = 0 or 2 partitioned to co-migrate with albumin, whereas amphiphiles with n>2 showed increasing fractions of the amphiphiles migrating as intact micelles in the presence of serum with increasing n. e, Fluorescein-labelled lipo-G6-CpG (5µM) and Alexa Fluor 647-labelled BSA (5µM) were incubated for 2h at 37°C in PBS plus 20mM KCl, and then analysed by SEC. Spectra were monitored at 480nm (ODN channel, green line, fluorescein) and 640nm (protein channel, red line, Alexa Fluor 647). The majority of BSA and CpG micellar aggregates eluted separately. f, Size of amph-CpG micelles as determined by dynamic light scattering. All data are mean±s.e.m. Statistical analysis was performed by one-way ANOVA with Bonferroni post-tests.

  7. LN localization of amph-CpGs with macrophages and dendritic cells.
    Extended Data Fig. 3: LN localization of amph-CpGs with macrophages and dendritic cells.

    a, Immunofluorescent images of inguinal LN section 24h after injection of 3.3nmol lipo-CpG or lipo-G2-CpG, showing dendritic cells (CD11c, blue), macrophages (F4/80, red) and CpG (green). bd, Mice (n = 3 per group) were injected subcutaneously with 3.3nmol of fluorescein-labelled CpG formulations. After 24h, LNs were digested and LN cells stained with 4′,6-diamidino-2-phenylindole (DAPI) and antibodies against F4/80, CD11c and CD207. b, c, Shown are representative flow cytometry plots of F4/80 staining (b) and CD11c staining (c) versus CpG fluorescence in viable (DAPI) cells. d, percentages of CpG+ cells in the LNs determined by flow cytometry at 24h. ***P<0.001; all data are mean±s.e.m. Statistical analysis was performed by unpaired Student’s t-test.

  8. CpG-albumin conjugates accumulate in LNs.
    Extended Data Fig. 4: CpG-albumin conjugates accumulate in LNs.

    a, b, C57BL/6 mice (n = 4 LNs per group) were injected subcutaneously at the tail base with 3.3nmol fluorescein-labelled free CpG, mouse albumin-CpG conjugates (MSA-CpG) or lipo-CpG. Inguinal LNs and axillary LNs were isolated 24h after injection and imaged (a) and quantified (b) by IVIS optical imaging. All data are mean±s.e.m. **P<0.01 by one-way ANOVA with Bonferroni post-test.

  9. In vitro characterization of amph-CpG.
    Extended Data Fig. 5: In vitro characterization of amph-CpG.

    a, Rhodamine-labelled CpG or amph-CpG (1μM) was incubated with murine bone-marrow-derived dendritic cells at 37°C for 4h with LysoTracker (Life Technologies) and imaged using a Zeiss LSM 510 confocal microscope. b, Rhodamine-labelled CpG or amph-CpG (1μM) was incubated for 30min, 2h, 6h and 24h with the murine dendritic cell line DC2.4. Cells were stained with DAPI and uptake was quantified by flow cytometry using the mean fluorescence intensity (MFI) of viable (DAPI) cells. c, Amph-CpG or PAM2CSK4 (a strong TLR2 agonist) was incubated for 24h with the InvivoGen HEK-Blue murine TLR2 reporter cell line, a secreted embryonic alkaline phosphatase (SEAP) reporter system. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1h and reading absorption at 620nm. d, Amph-CpG, CpG or control amph-GpC (1μM) were incubated with InvivoGen RAW-Blue mouse macrophage reporter cells, which secrete SEAP upon TLR, NOD or Dectin-1 stimulation. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1h and reading absorption at 620nm. e, Bone-marrow-derived immature dendritic cells were incubated overnight with indicated concentrations of OVA and maturation stimuli (or medium alone). Dendritic cells were washed three times with PBS and 30,000 CFSE-labelled OT-I CD8+ T cells were then added to each well. Cells were collected after 2 days of co-culture, and stained and gated for DAPI (viable) CD8+ T cells using Flowjo v.7.6.5 (Treestar). The extent of proliferation was quantified by determining the percentage of cells that had undergone division by determining the percentage of viable CD8+ T cells that had diluted CFSE using T cells alone as a control for the no division/dilution peak. Shown are mean±s.e.m. bd, ***P<0.001 by one-way ANOVA with Bonferroni post-test. Bars in e represent medians and whiskers represent range (n = 2 wells per condition).

  10. Albumin-binding lipo-CpGs elicit robust expansion of antigen-specific CD8+ T-cells when combined with soluble protein.
    Extended Data Fig. 6: Albumin-binding lipo-CpGs elicit robust expansion of antigen-specific CD8+ T-cells when combined with soluble protein.

    ah, Groups of C57BL/6 mice (n = 4–8 per group) were immunized subcutaneously on day 0 and day 14 with 10µg OVA and 1.24nmol CpG formulations as indicated. Six days after the final immunization, mice were bled and peripheral blood mononuclear cells were evaluated by SIINFEKL-tetramer staining and intracellular cytokine staining. a, Representative flow cytometric dot plots of H-2Kb/SIINFEKL tetramer staining of CD8+ cells. b, Representative flow dot plots of intracellular staining on CD8+ cells for IFN-γ and TNF-α after 6h ex vivo restimulation with SIINFEKL peptide. c, Serum samples were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for anti-OVA IgG (day 34). d, Mice were immunized on day 0 and day 14 with 1.24nmol lipo-G2-CpG mixed with 10µg SIV Gag protein, blood samples were collected and analysed by peptide-MHC tetramer staining for CD8+ T cells recognizing the immunodominant AL11 epitope of Gag. e, f, Anti-MSA and anti-OVA IgG (e, day 20) or IgM (f, day 20) were measured by ELISA. g, Groups of C57BL/6 mice (n = 4 per group) were immunized with 10µg OVA alone or mixed with 1.24nmol of a non-TLR agonist lipo-GpC, the same diacyl lipid tail conjugated to PEG (lipo-PEG, 48 EG units), or DSPE-PEG2000. Mice were boosted with the same formulation on day 14, and OVA tetramer+ CD8+ T cells in peripheral blood were assayed by flow cytometry on day 20. ***P<0.001. h, TLR2 knockout or wild-type mice were immunized as described in a, and OVA tetramer+ CD8+ T cells were assayed as previously. All data are mean±s.e.m. *P<0.05. Statistical analysis was performed by unpaired Student’s t-test.

  11. Albumin-binding CpG induces local lymphadenopathy but reduces systemic toxicity compared with soluble CpG adjuvant.
    Extended Data Fig. 7: Albumin-binding CpG induces local lymphadenopathy but reduces systemic toxicity compared with soluble CpG adjuvant.

    a, C57BL/6 mice (n = 3 per group) were injected with 1.24nmol CpGs subcutaneously on day 0 and 2.48nmol CpGs on days 2 and 4. On day 6 mice were killed and LNs were isolated and photographed with a digital camera. b, Bead-based flow analysis of proinflammatory cytokines elicited in peripheral blood of mice injected with a single dose (6.2nmol) of different CpG formulations. Blood samples were collected at different time intervals and analysed for TNF-α, as per manufacturer’s instructions. All data are mean±s.e.m. **P<0.01, *P<0.05. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test.

  12. Hydrophilic block length of amphiphiles determines cell membrane insertion and LN accumulation.
    Extended Data Fig. 8: Hydrophilic block length of amphiphiles determines cell membrane insertion and LN accumulation.

    a, Amphiphiles with varying hydrophilic PEG lengths were prepared by solid phase synthesis of diacyl tails coupled to 1–8 hexa-ethylene glycol phosphorothioate units. Fluorescein was incorporated either at the 3′ terminal (for membrane insertion analyses) or adjacent to the lipid moiety (for albumin-binding analyses). b, Splenocytes from C57BL/6 mice (5×107cells per ml) were incubated with lipo-(PEG)n-fluorescein (1.67μM) and albumin (100μM) at 37°C for 1h. Shown is a representative image of membrane insertion observed by confocal microcopy for lipo-(PEG)1-fluorescein. c, Equilibrium partitioning measurements shown as a function of albumin concentration at 37°C. Lipo-fluorescein-(PEG)n (5μM) was incubated with varying concentrations of BSA and fluorescence intensities were monitored by fluorescence spectroscopy. BSA binding disrupted the micellar structure and decreased the self-quenching of fluorescein. All samples reached maximum fluorescence intensities at around 10μM BSA, indicating 100% micelle breakup at this concentration (at higher BSA concentrations, fluorescence decreases due to solution turbidity). Arrow indicates plasma concentration of albumin. d, e, Lipo-Tn-FAM amphiphiles (n = 5, 10, 15, 20) were injected subcutaneously in C57BL/6 mice (n = 4 LNs per group), and excised LNs were imaged after 24h (d). Mean LN fluorescence from groups of mice are plotted in e. All data are mean±s.e.m.

  13. Lipo-PEG-peptide amphiphiles exhibit greatly enhanced LN accumulation compared with unmodified peptides.
    Extended Data Fig. 9: Lipo-PEG-peptide amphiphiles exhibit greatly enhanced LN accumulation compared with unmodified peptides.

    a, Peptides with amino-terminal cysteines were conjugated to maleimide-PEG2000-DSPE. b, c, FAM-labelled immunodominant peptide derived from the HPV-16 E7 protein (FAM-FTVINYHARC, synthesized in reverse sequence order using d-amino acids to obtain the same chiral organization of side chains as the typical l-amino acid sequence in a protease-resistant peptide) was injected subcutaneously at the tail base as a free peptide (D-E7) or as a PEG-DSPE conjugate (amph-D-E7). Shown are IVIS images of draining LNs 24h after injection (b) and fluorescence quantifications (c). All data are mean±s.e.m. **P<0.01. Statistical analysis was performed by unpaired Student’s t-test.

  14. Long-peptide amphiphiles, when combined with amph-CpG, elicit a potent antigen-specific CD8+ T-cell response with therapeutic benefits, as compared to soluble formulation.
    Extended Data Fig. 10: Long-peptide amphiphiles, when combined with amph-CpG, elicit a potent antigen-specific CD8+ T-cell response with therapeutic benefits, as compared to soluble formulation.

    a, C57BL/6 mice were primed on day 0 and boosted on day 14 with amph-E7long (HPV-16 E743–62, 10μg peptide) and amph-CpG (lipo-G2-CpG, 1.24nmol), or equivalent soluble peptide/CpG vaccines. Six days after the boost, mice were bled and analysed for tetramer-positive CD8+ T cells in peripheral blood. be, C57BL/6 mice (n = 8 per group) were inoculated with 3×105 TC-1 tumour cells subcutaneously in the flank and left untreated or immunized with soluble or amphiphile long-peptide vaccines on days 6 (10µg peptide, 1.24nmol CpG), 13 (20µg peptide, 1.24nmol CpG) and 19 (20µg peptide, 1.24nmol CpG). bd, Shown are individual tumour growth curves for no treatment (b), immunization with soluble E7long and CpG (c), or immunization with amph-E7long plus amph-CpG (d). Kaplan–Meier survival curves of eight mice per group are shown in e. f, Long-peptide amphiphiles also elicit potent immune responses when combined with non-CpG, non-LN-targeting alternative adjuvants. C57BL/6 mice (n = 4 per group) were immunized as before, using amph-E7long peptide (10μg) combined with monophosphoryl lipid A (MPLA, 10μg) or polyinosinic:polycytidylic acid (poly I:C, 50μg). The frequencies of E7 tetramer+ CD8+ T cells in peripheral blood were assayed on day 20. All data are mean±s.e.m. ***P<0.001, **P<0.01, *P<0.05 by unpaired Student’s t-test.

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Author information

Affiliations

  1. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Haipeng Liu &
    • Darrell J. Irvine
  2. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Haipeng Liu,
    • Kelly D. Moynihan,
    • Yiran Zheng,
    • Gregory L. Szeto,
    • Adrienne V. Li,
    • Bonnie Huang,
    • Clara Park &
    • Darrell J. Irvine
  3. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Haipeng Liu,
    • Kelly D. Moynihan,
    • Yiran Zheng,
    • Gregory L. Szeto,
    • Adrienne V. Li,
    • Bonnie Huang &
    • Darrell J. Irvine
  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Debra S. Van Egeren
  5. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Cambridge, Massachusetts 02139, USA

    • Darrell J. Irvine
  6. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA

    • Darrell J. Irvine

Contributions

H.L. designed and performed most experiments and analysed the data, and wrote the manuscript; Y.Z. carried out tumour therapy experiments and analysed the data. K.D.M. carried out in vitro bioactivity studies of CpG, biolayer interforometry binding studies and in vivo immunizations of SIV Gag and analysed the data. A.V.L. and B.H. assisted in tetramer/in vivo cytotoxicity assays and contributed experimental suggestions. G.L.S. assisted with optimization of proinflamatory cytokine assays and helped in vitro bioactivity studies of CpG. G.L.S., C.P. and D.S.V.E. contributed to in vitro T-cell proliferation assays. D.J.I. supervised all experiments and wrote the manuscript.

Competing financial interests

A patent application for amphiphile vaccines has been filed, with D.J.I. and H.L. as inventors.

Corresponding author

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Interaction between albumin and amph-CpGs. (297 KB)

    a, SEC of FBS, albumin and fluorescein-labelled amph-CpGs. FBS and bovine serum albumin (BSA) were monitored using absorptions at 280nm, whereas CpG oligonucleotides were monitored at 480nm (fluorescein peak). b, Lipo-CpG, but not CpG, interacts with serum albumin as shown by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) after protein pull-down assays: FBS was incubated with 3′-biotin-labelled CpG (CpG-biotin), lipo-CpG (lipo-CpG-biotin) or PBS for 1h at 37°C. Streptavidin-conjugated magnetic beads were added to capture biotinylated CpGs and any associated proteins, separated by a magnet, boiled to release bound CpG/proteins, and subjected to SDS–PAGE analysis. Lane 1: protein MW ladder; lane 2: purified BSA; lane 3: FBS (100× dilution, 10μl loading); lane 4: pull-down control, FBS incubated with streptavidin-magnetic beads; lane 5: pull-down with CpG-biotin, FBS was incubated with CpG-biotin and streptavidin magnetic beads; lane 6: pull-down with lipo-CpG-biotin, FBS incubated with lipo-CpG-biotin and streptavidin magnetic beads; lane 7: FBS (100× dilution, 5μl loading). c, Fluorescein-labelled CpG or lipo-CpG was incubated with albumin-conjugated agarose resin for 1h at 37°C, and the resin was separated by filtration. The filtrate and recovered agarose were visualized by a gel imager and quantified by fluorescence measurements. d, Bio-layer interferometry measurements of lipo-CpG and CpG binding to immobilized BSA. Albumin-conjugated BLI probes were immersed in solutions of lipo-CpG, and wavelength shifts (Δλ) of the interferometry pattern association and dissociation curves were followed over time to determine affinity constants of binding at 25°C. Shown are apparent kon, koff and KD values from fits to the data. e, FRET between FAM-labelled lipo-CpG and rhodamine-conjugated albumin (BSA-Rh) assessed by fluorescence spectroscopy. CpG-F or lipo-CpG-F (1.65µM) alone or mixed with BSA-Rh (1.5µM) in PBS were excited at 488nm and emission was recorded from 500–650nm.

  2. Extended Data Figure 2: Construction and characterization of G-quadruplex-stabilized CpG adjuvants. (641 KB)

    a, G-quadruplex-stabilized CpG micelles are self-assembled from amphiphiles composed of three distinct segments: an immunostimulatory CpG sequence, a central repeat block containing n = 1–10 G-quartet-forming guanines followed by (10n) non-interacting thymidines, and a diacyl lipid tail. In aqueous solutions, these amphiphiles self-assembled into three-dimensional spherical micelles with a CpG corona and a lipid core. In the presence of K+, neighbouring guanine repeats in the oligonucleotide corona form G-quadruplex structures via Hoogsteen hydrogen bonds and stabilize the micelle structure. The stability of the oligonucleotide micelles in the presence of serum was programmed by altering the length of the guanine repeat. b, Parallel G-quartet formation among DNA strands within the micelles was detected by circular dichroism (CD) spectroscopy, as manifested by the shifting of positive peaks from 278nm towards 262nm and troughs at 245nm as the number of guanines in the structure increased. c, Pyrene excimer fluorescence was used to assay the stabilities of G-quadruplex micelles in the presence of albumin: pyrene dye incorporated in stabilized CpG micelles (n>2) retained excimer fluorescence in the presence of high concentrations of albumin. By contrast, albumin binds to the lipid moiety of unstabilized micelles (n2) and disrupts the micelle structures, leading to loss of excimer fluorescence in an albumin-concentration-dependent manner as the protein disrupts the micelles into albumin-bound unimers. Shown below the schematic is the fraction of amph-CpG remaining in the micellar state as a function of albumin concentration as reported by excimer fluorescence. Arrow indicates the plasma concentration of albumin. d, Stability profiles of G-quadruplex CpG micelles as measured by SEC in the presence of FBS. Fluorescein-labelled CpG micelles were incubated with 20% FBS in PBS in the presence of 10mM Mg2+ and 20mM K+ at 37°C for 2h, then analysed by SEC. FBS and BSA were monitored using absorptions at 280nm, whereas lipo-Gn-CpG amphiphiles were monitored at 480nm (fluorescein peak). Lipo-Gn-CpG with n = 0 or 2 partitioned to co-migrate with albumin, whereas amphiphiles with n>2 showed increasing fractions of the amphiphiles migrating as intact micelles in the presence of serum with increasing n. e, Fluorescein-labelled lipo-G6-CpG (5µM) and Alexa Fluor 647-labelled BSA (5µM) were incubated for 2h at 37°C in PBS plus 20mM KCl, and then analysed by SEC. Spectra were monitored at 480nm (ODN channel, green line, fluorescein) and 640nm (protein channel, red line, Alexa Fluor 647). The majority of BSA and CpG micellar aggregates eluted separately. f, Size of amph-CpG micelles as determined by dynamic light scattering. All data are mean±s.e.m. Statistical analysis was performed by one-way ANOVA with Bonferroni post-tests.

  3. Extended Data Figure 3: LN localization of amph-CpGs with macrophages and dendritic cells. (729 KB)

    a, Immunofluorescent images of inguinal LN section 24h after injection of 3.3nmol lipo-CpG or lipo-G2-CpG, showing dendritic cells (CD11c, blue), macrophages (F4/80, red) and CpG (green). bd, Mice (n = 3 per group) were injected subcutaneously with 3.3nmol of fluorescein-labelled CpG formulations. After 24h, LNs were digested and LN cells stained with 4′,6-diamidino-2-phenylindole (DAPI) and antibodies against F4/80, CD11c and CD207. b, c, Shown are representative flow cytometry plots of F4/80 staining (b) and CD11c staining (c) versus CpG fluorescence in viable (DAPI) cells. d, percentages of CpG+ cells in the LNs determined by flow cytometry at 24h. ***P<0.001; all data are mean±s.e.m. Statistical analysis was performed by unpaired Student’s t-test.

  4. Extended Data Figure 4: CpG-albumin conjugates accumulate in LNs. (202 KB)

    a, b, C57BL/6 mice (n = 4 LNs per group) were injected subcutaneously at the tail base with 3.3nmol fluorescein-labelled free CpG, mouse albumin-CpG conjugates (MSA-CpG) or lipo-CpG. Inguinal LNs and axillary LNs were isolated 24h after injection and imaged (a) and quantified (b) by IVIS optical imaging. All data are mean±s.e.m. **P<0.01 by one-way ANOVA with Bonferroni post-test.

  5. Extended Data Figure 5: In vitro characterization of amph-CpG. (345 KB)

    a, Rhodamine-labelled CpG or amph-CpG (1μM) was incubated with murine bone-marrow-derived dendritic cells at 37°C for 4h with LysoTracker (Life Technologies) and imaged using a Zeiss LSM 510 confocal microscope. b, Rhodamine-labelled CpG or amph-CpG (1μM) was incubated for 30min, 2h, 6h and 24h with the murine dendritic cell line DC2.4. Cells were stained with DAPI and uptake was quantified by flow cytometry using the mean fluorescence intensity (MFI) of viable (DAPI) cells. c, Amph-CpG or PAM2CSK4 (a strong TLR2 agonist) was incubated for 24h with the InvivoGen HEK-Blue murine TLR2 reporter cell line, a secreted embryonic alkaline phosphatase (SEAP) reporter system. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1h and reading absorption at 620nm. d, Amph-CpG, CpG or control amph-GpC (1μM) were incubated with InvivoGen RAW-Blue mouse macrophage reporter cells, which secrete SEAP upon TLR, NOD or Dectin-1 stimulation. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1h and reading absorption at 620nm. e, Bone-marrow-derived immature dendritic cells were incubated overnight with indicated concentrations of OVA and maturation stimuli (or medium alone). Dendritic cells were washed three times with PBS and 30,000 CFSE-labelled OT-I CD8+ T cells were then added to each well. Cells were collected after 2 days of co-culture, and stained and gated for DAPI (viable) CD8+ T cells using Flowjo v.7.6.5 (Treestar). The extent of proliferation was quantified by determining the percentage of cells that had undergone division by determining the percentage of viable CD8+ T cells that had diluted CFSE using T cells alone as a control for the no division/dilution peak. Shown are mean±s.e.m. bd, ***P<0.001 by one-way ANOVA with Bonferroni post-test. Bars in e represent medians and whiskers represent range (n = 2 wells per condition).

  6. Extended Data Figure 6: Albumin-binding lipo-CpGs elicit robust expansion of antigen-specific CD8+ T-cells when combined with soluble protein. (318 KB)

    ah, Groups of C57BL/6 mice (n = 4–8 per group) were immunized subcutaneously on day 0 and day 14 with 10µg OVA and 1.24nmol CpG formulations as indicated. Six days after the final immunization, mice were bled and peripheral blood mononuclear cells were evaluated by SIINFEKL-tetramer staining and intracellular cytokine staining. a, Representative flow cytometric dot plots of H-2Kb/SIINFEKL tetramer staining of CD8+ cells. b, Representative flow dot plots of intracellular staining on CD8+ cells for IFN-γ and TNF-α after 6h ex vivo restimulation with SIINFEKL peptide. c, Serum samples were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for anti-OVA IgG (day 34). d, Mice were immunized on day 0 and day 14 with 1.24nmol lipo-G2-CpG mixed with 10µg SIV Gag protein, blood samples were collected and analysed by peptide-MHC tetramer staining for CD8+ T cells recognizing the immunodominant AL11 epitope of Gag. e, f, Anti-MSA and anti-OVA IgG (e, day 20) or IgM (f, day 20) were measured by ELISA. g, Groups of C57BL/6 mice (n = 4 per group) were immunized with 10µg OVA alone or mixed with 1.24nmol of a non-TLR agonist lipo-GpC, the same diacyl lipid tail conjugated to PEG (lipo-PEG, 48 EG units), or DSPE-PEG2000. Mice were boosted with the same formulation on day 14, and OVA tetramer+ CD8+ T cells in peripheral blood were assayed by flow cytometry on day 20. ***P<0.001. h, TLR2 knockout or wild-type mice were immunized as described in a, and OVA tetramer+ CD8+ T cells were assayed as previously. All data are mean±s.e.m. *P<0.05. Statistical analysis was performed by unpaired Student’s t-test.

  7. Extended Data Figure 7: Albumin-binding CpG induces local lymphadenopathy but reduces systemic toxicity compared with soluble CpG adjuvant. (176 KB)

    a, C57BL/6 mice (n = 3 per group) were injected with 1.24nmol CpGs subcutaneously on day 0 and 2.48nmol CpGs on days 2 and 4. On day 6 mice were killed and LNs were isolated and photographed with a digital camera. b, Bead-based flow analysis of proinflammatory cytokines elicited in peripheral blood of mice injected with a single dose (6.2nmol) of different CpG formulations. Blood samples were collected at different time intervals and analysed for TNF-α, as per manufacturer’s instructions. All data are mean±s.e.m. **P<0.01, *P<0.05. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test.

  8. Extended Data Figure 8: Hydrophilic block length of amphiphiles determines cell membrane insertion and LN accumulation. (275 KB)

    a, Amphiphiles with varying hydrophilic PEG lengths were prepared by solid phase synthesis of diacyl tails coupled to 1–8 hexa-ethylene glycol phosphorothioate units. Fluorescein was incorporated either at the 3′ terminal (for membrane insertion analyses) or adjacent to the lipid moiety (for albumin-binding analyses). b, Splenocytes from C57BL/6 mice (5×107cells per ml) were incubated with lipo-(PEG)n-fluorescein (1.67μM) and albumin (100μM) at 37°C for 1h. Shown is a representative image of membrane insertion observed by confocal microcopy for lipo-(PEG)1-fluorescein. c, Equilibrium partitioning measurements shown as a function of albumin concentration at 37°C. Lipo-fluorescein-(PEG)n (5μM) was incubated with varying concentrations of BSA and fluorescence intensities were monitored by fluorescence spectroscopy. BSA binding disrupted the micellar structure and decreased the self-quenching of fluorescein. All samples reached maximum fluorescence intensities at around 10μM BSA, indicating 100% micelle breakup at this concentration (at higher BSA concentrations, fluorescence decreases due to solution turbidity). Arrow indicates plasma concentration of albumin. d, e, Lipo-Tn-FAM amphiphiles (n = 5, 10, 15, 20) were injected subcutaneously in C57BL/6 mice (n = 4 LNs per group), and excised LNs were imaged after 24h (d). Mean LN fluorescence from groups of mice are plotted in e. All data are mean±s.e.m.

  9. Extended Data Figure 9: Lipo-PEG-peptide amphiphiles exhibit greatly enhanced LN accumulation compared with unmodified peptides. (242 KB)

    a, Peptides with amino-terminal cysteines were conjugated to maleimide-PEG2000-DSPE. b, c, FAM-labelled immunodominant peptide derived from the HPV-16 E7 protein (FAM-FTVINYHARC, synthesized in reverse sequence order using d-amino acids to obtain the same chiral organization of side chains as the typical l-amino acid sequence in a protease-resistant peptide) was injected subcutaneously at the tail base as a free peptide (D-E7) or as a PEG-DSPE conjugate (amph-D-E7). Shown are IVIS images of draining LNs 24h after injection (b) and fluorescence quantifications (c). All data are mean±s.e.m. **P<0.01. Statistical analysis was performed by unpaired Student’s t-test.

  10. Extended Data Figure 10: Long-peptide amphiphiles, when combined with amph-CpG, elicit a potent antigen-specific CD8+ T-cell response with therapeutic benefits, as compared to soluble formulation. (325 KB)

    a, C57BL/6 mice were primed on day 0 and boosted on day 14 with amph-E7long (HPV-16 E743–62, 10μg peptide) and amph-CpG (lipo-G2-CpG, 1.24nmol), or equivalent soluble peptide/CpG vaccines. Six days after the boost, mice were bled and analysed for tetramer-positive CD8+ T cells in peripheral blood. be, C57BL/6 mice (n = 8 per group) were inoculated with 3×105 TC-1 tumour cells subcutaneously in the flank and left untreated or immunized with soluble or amphiphile long-peptide vaccines on days 6 (10µg peptide, 1.24nmol CpG), 13 (20µg peptide, 1.24nmol CpG) and 19 (20µg peptide, 1.24nmol CpG). bd, Shown are individual tumour growth curves for no treatment (b), immunization with soluble E7long and CpG (c), or immunization with amph-E7long plus amph-CpG (d). Kaplan–Meier survival curves of eight mice per group are shown in e. f, Long-peptide amphiphiles also elicit potent immune responses when combined with non-CpG, non-LN-targeting alternative adjuvants. C57BL/6 mice (n = 4 per group) were immunized as before, using amph-E7long peptide (10μg) combined with monophosphoryl lipid A (MPLA, 10μg) or polyinosinic:polycytidylic acid (poly I:C, 50μg). The frequencies of E7 tetramer+ CD8+ T cells in peripheral blood were assayed on day 20. All data are mean±s.e.m. ***P<0.001, **P<0.01, *P<0.05 by unpaired Student’s t-test.

Supplementary information

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  1. Supplementary Information (310 KB)

    This file contains Supplementary Methods, Supplementary Table 1 and Supplementary References.

Additional data