A biomimetic multi-component subunit vaccine via ratiometric loading of hierarchical hydrogels

Abstract

The development of subunit vaccines that mimic the molecular complexity of attenuated vaccines has been limited by the difficulty of intracellular co-delivery of multiple chemically diverse payloads at controllable concentrations. We report on hierarchical hydrogel depots employing simple poly(propylene sulfone) homopolymers to enable ratiometric loading of a protein antigen and four physicochemically distinct adjuvants in a hierarchical manner. The optimized vaccine consisted of immunostimulants either adsorbed to or encapsulated within nanogels, which were capable of noncovalent anchoring to subcutaneous tissues. In female BALB/c and C57BL/6 mice, these 5-component nanogel vaccines demonstrated enhanced humoral and cell-mediated immune responses compared to formulations with standard single adjuvant and antigen pairing. The use of a single simple homopolymer capable of rapid and stable loading and intracellular delivery of diverse molecular cargoes holds promise for facile development and optimization of scalable subunit vaccines and complex therapeutic formulations for a wide range of biomedical applications.

Fig. 1. Schematic of PPSU-based hierarchical hydrogels that enable ratiometric loading of multiple adjuvants for vaccine optimization.

Hydrophobic adjuvants are encapsulated through network assembly of PPSU (1^st gelation). Water-soluble adjuvants are adsorbed during the formation of microgels triggered by bridging nanogels with the antigen OVA (2^nd gelation, only simple mixing steps that take less than 5 min before injection). Microgels anchor onto collagens upon subcutaneous injection (3^rd gelation), allowing sustained release. Within the first 24 h after vaccination, Th1/Th2 cytokine levels increased. By day 7, anti-OVA IgM antibodies had risen, and over the subsequent four days, the vaccination effectively promoted the proliferation of adoptively transferred antigen-specific T cells. Anti-OVA IgG2b and IgG2c responses remained significantly elevated for up to one month, while significant levels of anti-OVA IgG1 antibodies persisted through day 84. Following a prime-boost administration on day 90 and a subsequent 15-day period, marked increases in neutrophils, CD11b⁺ conventional dendritic cells (cDCs), and B cells were observed.

Fig. 2. PPSU nanogels enable the ratiometric loading of multiple cargoes and subsequent assembly into microgels.

a Comparison of the extinction spectra of the mixture of PPSU nanogels (1 mg/mL) with 1 wt.% of OVA in PBS or water. b CryoSEM of PPSU microgels following salt-induced gelation of nanogels via OVA bridges in PBS. c CryoTEM of PPSU microgels formed by mixing PPSU nanogels with OVA in PBS. (b-c) All experiments were independently repeated three times. d The complete self-quenching indicates the efficient capture of ICG during the formation of microgels in PBS. The inset shows ICG/PBS solutions before and after the addition of PPSU nanogels. The photos were taken after centrifugation. e Computational studies suggest that the PPSU surface is capable of molecular capture through hydrophobic interactions (Lennard-Jones). f Multiple adjuvants, including MPLA, CL420, CpG, and LPS can be co-loaded in a ratiometric manner while using the antigen OVA to bridge nanogels.

Fig. 3. Characterization of tissue-bound hydrogels that formed following subcutaneous injection of PPSU microgels.

a Dropping PPSU microgels (colored by ICG) onto an excised skin mimics the process of subcutaneous injection, anchoring the microgels onto tissues. b Frequency-dependent oscillatory experiments conducted in a linear viscoelastic regime. c The storage and loss moduli across the evaluated frequency range. (b-c) Rheological measurements were performed on excised samples 30 min post injection, demonstrating the in-situ formation of tertiary hydrogels. d SEM images of excised gels on day 0 (30 min post injection) and 5 days post injection. The SEM image of collagen fibers is included for comparison (ctr = control). e EDS elemental mapping of Fe³⁺-loaded hydrogels 30 min post injection. d, e All experiments were independently repeated three times.

Fig. 4. In vivo release of model cargoes loaded within PPSU microgels.

a Real-time whole-body imaging of adsorbed ICG after SC injection showed prolonged release of hydrogel cargo. The data are presented as mean values with the statistical significance determined by a two-sample t-test (n = 3 mice per group). b Efficient FRET demonstrates full accessibility (within ~10 nm) of the microgel surfaces by small molecule fluorophore Rh101. c The recovery of Rh6G fluorescence at day 1 indicated quick desorption/replacement of adsorbed Rh101 upon SC injection. λ_em/λ_ex = 465/640 nm for Rh101, λ_em/λ_ex = 465/560 nm for Rh6G. The data are presented as mean values with the statistical significance determined by a two-sample t-test (n = 5 mice per group). d Not all of the microgel surfaces are accessible to proteins, as suggested by the inefficient FRET from encapsulated MFT in response to incubation with TNF-α. e Sustained release was achieved for the encapsulated MFT and adsorbed proteins. λ_em/λ_ex = 465/600 nm for TNF-α, λ_em/λ_ex = 465/520 nm for MFT. The data are presented as mean values with n = 3 mice per group.

Fig. 5. Validation of multi-adjuvant/antigen-loaded PPSU hydrogels as a subunit vaccine.

a Schematic schedule of in vivo blood analysis and adoptive T cell transfer. (created with BioRender.com). b Cytokine fold changes 24 h after subcutaneous injection of the multi-antigen/adjuvant (LPS excluded) loaded PPSU relative to PBS control in mice. n = 3 mice per group (mean ± s.d). c Blood anti-OVA IgM antibody levels 7 days post subcutaneous administration of the multi-antigen/adjuvant (LPS excluded) loaded PPSU relative to single adjuvant, blank PPSU and PBS controls. n = 3 mice per group (mean ± s.d), with *p (0.0355) < 0.05, **p (0.0034) < 0.01, determined using one-way ANOVA followed by Tukey’s multiple comparison test. d Flow cytometry histogram and e mean fluorescence intensity (MFI) of activation markers (MHC-II, CD40, CD80, and CD86) on dendritic cells in the lymph node after 4 days following the adoptive transfer of carboxyfluorescein succinimidyl ester (CFSE) stained CD8⁺ T cell from OT-1 mice to C57BL/6 mice. n  =  4 mice per group (mean ± s.d). *p (0.0411, MHC-II), p (0.044, CD40), p (0.0193, CD80), p (0.0393, CD86) < 0.05, **p (0.0099, MHC-II), p (0.0092, CD40), p (0.0067, CD86) < 0.01, and ns: not significant by unpaired two‑tailed t-test. f Gating strategies and percentage of live CFSE⁺CD8⁺ T cells at lymph node. n  =  4 mice per group (mean ± s.d). *p (0.0127, and 0.0169) < 0.05 by unpaired two‑tailed t-test. and g spleen after 4 days post adoptive T cell transfer. n  =  4 mice per group (mean ± s.d). **p (0.0064) < 0.01, and ns not significant by unpaired two‑tailed t-test.

Fig. 6. Long-term presentation of subunit PPSU vaccines after a single administration.

a Experimental timeline: Antibody concentrations were measured after a single subcutaneous injection of either a bolus formulation (100 μg OVA, 20 μg CpG, 25 μg MPLA, 25 μg CL429, and 25 μg LPS) or PPSU vaccines containing one (PPSU-LPS) or four adjuvants (PPSU-4Ad). Measurements began on day 7 and continued until day 84. b Serum anti-OVA IgG1 concentrations: (n = 6 animals per group; samples pooled and measured in triplicate). Data are presented as mean ± S.D., with **** p < 0.0001 compared to bolus and PPSU1, determined using two-way ANOVA followed by Sidak’s multiple comparison test. c Mouse body weight changes: Weight changes were monitored after vaccination (n = 6 animals per group). Data are presented as mean ± S.D. d, e Serum anti-OVA IgG2b and IgG2c concentrations: These were evaluated on day 28 post-vaccination (n = 6 animals per group; samples pooled and measured in triplicate). Data are presented as mean ± S.D. ns: no significant difference compared to bolus; **p (0.0013, 0.0036, IgG2b), p (0.0019, IgG2c) < 0.01 compared to bolus, determined using one-way ANOVA followed by Tukey’s multiple comparison test. f–h Flow cytometry analysis of CD45⁺ splenic cell populations 2 weeks post-boost (n = 6 animals per group; data presented as mean ± S.D). ns no significant difference compared to bolus; **p (0.0058, B cells) <0.01 compared to bolus, **** p < 0.0001 compared to bolus, determined using one-way ANOVA followed by Tukey’s multiple comparison test: f CD11b⁺ conventional dendritic cells (cDCs), g Neutrophils, and h B cells.