Modulation of injectable hydrogel properties for slow co‐delivery of influenza subunit vaccine components enhance the potency of humoral immunity

Abstract Vaccines are critical for combating infectious diseases across the globe. Influenza, for example, kills roughly 500,000 people annually worldwide, despite annual vaccination campaigns. Efficacious vaccines must elicit a robust and durable antibody response, and poor efficacy often arises from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow‐delivery influenza vaccine platform. We utilized an injectable and self‐healing polymer‐nanoparticle (PNP) hydrogel platform to prolong the co‐delivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique dynamic physical characteristics whereby physicochemically distinct influenza hemagglutinin antigen and a toll‐like receptor 7/8 agonist adjuvant could be co‐delivered over prolonged timeframes that were tunable through simple alteration of the gel formulation. We show a relationship between hydrogel physical properties and the resulting immune response to immunization. When administered in mice, hydrogel‐based vaccines demonstrated enhancements in the magnitude and duration of humoral immune responses compared to alum, a widely used clinical adjuvant system. We found stiffer hydrogel formulations exhibited slower release and resulted in the greatest improvements to the antibody response while also enabling significant adjuvant dose sparing. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

immunization. Numerous efforts have been taken to engineer novel vaccination approaches to promote higher degrees of affinity maturation or to target highly conserved epitopes by engineering multivalent antigen nanoparticles 4,5 or novel antigen proteins. 6,7 In addition to antigen engineering, many efforts have focused on the development of more potent adjuvants, such as toll-like receptor agonists (TLRa) to improve the immunogenicity of the subunit antigens. Unfortunately, while reports indicate that subunit vaccines comprising multiple TLRa molecules elicit better immune memory and stronger antibody responses, [8][9][10] it was recently reported that numerous highly potent adjuvant systems increase binding antibody titer but do not affect levels of somatic hypermutation when used in HIV vaccines in non-human primates. 11 These results indicate that adjuvants alone, even potent TLRa adjuvants, may not be sufficient to spur high quality immune responses, highlighting that a critical need exists for improved spatiotemporal control over vaccine delivery.
Further vaccine engineering has demonstrated that time-controlled release is favorable for eliciting stronger immune system responses. 12 It has recently been demonstrated that sustained release of an HIV vaccine using a surgically implantable osmotic pump leads to more robust and durable GC responses, higher antibody titers, and the targeting of a more diverse set of epitopes and better virus neutralization than standard bolus administration of the same vaccine. 13 While this work clearly highlighted the importance of sustained vaccine exposure on improving humoral immune responses, the pump system is cumbersome, and its implantation is invasive. Injectable depot technologies represent a promising, minimally-invasive approach to prolonged vaccine delivery. 14,15 Our group previously reported the use of dynamic, injectable polymer nanoparticle (PNP) hydrogel platform for prolonged co-delivery of subunit vaccine components to enhance humoral immune responses. 14,16 These PNP hydrogels consist of polymer strands dynamically cross-linked by multivalent non-covalent interactions with nanoparticles (NPs), allowing for dynamic detachment and reattachment to achieve injectability followed by robust depot formation. 17 This work reported that physicochemically distinct subunit vaccine cargo ovalbumin and poly(I:C), a potent TLR3a, could be co-delivered over prolonged time-frames, enhancing the magnitude and duration of GC responses and antibody responses, leading to a 1,000-fold increase in antibody affinity maturation. 14 To achieve potent, durable, and high-quality antibody responses to influenza vaccines, it appears that three parameters are necessary: (a) sustained vaccine exposure to prolong affinity maturation, (b) the use of adjuvants, and (c) precise co-delivery of subunit vaccine components. Currently, the relationship between timeframe of vaccine exposure and the resulting immune response is poorly understood.
Moreover, while numerous approaches have been shown to improve antigen delivery, [18][19][20][21] often the challenge of sustained co-delivery of antigen and adjuvant persists. A platform that allows for controlled release of multiple components is necessary to elicit potent responses. Yet, controlled encapsulation and sustained release of reagents differing in chemistry and size has proven challenging. 8 Indeed, many studies evaluating combinations of adjuvants have focused on those of similar physicochemical properties, such as CpG (TLR9a) and poly(I:C) (TLR3a) since they are both nucleic acid polymers or MPL (TLR4a) and imidazoquinolines (TLR7a or TLR7/8a) since they are both hydrophobic, and new materials technologies are required to generate opportunities to evaluate novel, synergistic adjuvant pairings 22,23 and improve subunit vaccine delivery.
When considering the use of hydrogels as delivery vehicles for subunit vaccines, it is of utmost importance to evaluate the mechanical properties of the gel. While previous works acted as a proof of concept for the use of PNP hydrogels as a vaccine delivery platform, 24 the relationship between material properties and the resulting immune response was not evaluated. Alteration of the hydrogel formulation will, for example, affect the diffusivity of vaccine cargo, thereby changing the timeframe of vaccine exposure. Additionally, the conjugation of TLR7/8a to the PEG-PLA NPs that act as a structural motif within the hydrogel network allows this small molecule adjuvant to diffuse at the same rate as an entrapped HA antigen protein. By tuning the diffusion kinetics of these cargo, we have shown it is possible to optimize the hydrogel formulation to achieve temporal and spatial co-delivery of adjuvant and influenza antigen.
In this work, we have sought to engineer PNP hydrogels to improve humoral immune response to influenza vaccines comprising HA and a potent TLR7/8a adjuvant, (1-(4-[aminomethyl]benzyl)-2-(ethoxymethyl)-1H-imidazo [4,5-c]quinolin-4-amine) (Figure 1), which is a derivative of Resiquimod (R848). We build on recent work where we demonstrated that by chemically conjugating the TLR7/8a to the NP structural motif of these hydrogels it is possible to achieve sustained antigen and adjuvant co-delivery. 24 We show that modulation of the PNP hydrogel formulation affects the structural and mechanical properties of the hydrogel, imparting control over cargo diffusion and achievable timescales of vaccine cargo co-delivery.
When administered in mice, hydrogel-based vaccines demonstrate enhancements in the magnitude and duration of humoral immune responses compared to alum, a widely used clinical adjuvant system. We find stiffer hydrogel formulations exhibiting slower release yield the greatest improvements to the antibody response while also enabling significant adjuvant dose sparing. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines. Aldrich and used as received. Monomethoxy-PEG (5 kDa) was purchased from Sigma Aldrich and was purified by azeotropic distillation with toluene prior to use. AF647-DBCO was purchased from Thermo Fisher and used as received. HIS-Lite-Cy3 Bis NTA-Ni Complex was purchased from AAT Bioquest and used as received. Cyanine3 NHS ester was purchased from Lumiprobe and used as received. F I G U R E 1 Fabrication of Polymer-Nanoparticle (PNP) Hydrogel and TLR7/8a Nanoparticles. (a) PNP hydrogels are formed when (i) poly (ethylene glycol)-b-poly(lactic acid) (PEG-PLA) nanoparticles (NPs) or TLR7/8 agonist (TLR7/8a) conjugated PEG-PLA NPs are combined with (ii) dodecyl-modified hydroxypropylmethylcellulose (HPMC-C 12 ). Multivalent and dynamic non-covalent interactions between the polymer and NPs constitute physical cross-links within the hydrogel structure. Vaccine cargo can be added to the aqueous NP solution before mixing. (b) NHS coupling of TLR7/8a to alkyne (I), and the coupling to azide-terminated PEG-PLA (II) to make PEG-PLA with the TLR7/8a (pink) presenting on the PEG (dark green) terminus of the block copolymer (III). This polymer can then be mixed with standard PEG-PLA and then nanoprecipitated into water to form TLR7/8a-NPs with a TLR7/8a valency defined by the physical mixture of the two polymers. (c) Gel formulations were varied by (i) changing the ratio of polymer: NP: solvent where 2:10 denotes a formulation comprising 2 wt% HPMC-C 12 : 10 wt% NP: 88 wt% solvent, (ii) altering the TLR7/8a-NP valency, and (iii) altering the total dose of entrapped TLR7/8a molecules consisted of an Optilab T-rEX (Wyatt Technology Corporation) refractive index detector operating at 658 nm and a HELEOS II light scattering detector (Wyatt Technology Corporation) operating at 659 nm. Dn/dc values for PEG and PLA were 0.0442 and 0.019 in the mobile phase were calculated using (dn/dc) ab = (dn/dc) a (wt %)a + (dn/dc) b (wt %) b after determining the dn/dc values for PEG and PEG-PLA polymers of known weight fractions (via 1 H-NMR spectroscopy) in the ASTRA software package by batch injection of three samples of known concentrations into an Optilab T-rEX refractive index detector.

| Preparation of HPMC-C 12
HPMC-C 12 was prepared according to previously reported procedures, 13,14 and the protocols will be briefly described here.
HPMC (1.0 g) was dissolved in NMP (40 ml) by stirring at 80 C for 1 h before removing the heat. Once the solution cooled to room temperature, 1-dodecylisocynate (105 mg, 0.5 mmol) and Hunig's base (catalyst, $3 drops) were dissolved in NMP (5.0 ml). This solution was added dropwise to the reaction mixture, which was then stirred at room temperature for 16 h. This solution was then precipitated from acetone, decanted, re-dissolved in water ($2 wt%) and placed in a dialysis tube for dialysis against water for 3-4 days. The polymer was lyophilized and reconstituted to a 60 mg/ml solution with sterile PBS.

| Preparation of PEG-PLA
PEG-PLA was prepared as previously reported, 25  2.6 | Preparation of TLR7/8a-PEG-PLA TLR7/8a-PEG-PLA was prepared according to a literature report 26 and the protocols will be briefly described here. Azide-PEG-PLA was prepared by organocatalytic ring opening polymerization using N 3 -PEG-OH (0.5 g, 5 kDa, 100 μmol) in anhydrous DCM (2.0 ml) with DBU (30 μl, 30 mg, 0.20 mmol) which was added quickly to a stirring solution of LA (2.0 g, 13.9 mmol) in anhydrous DCM (6.0 ml). The solution was stirred for 10 min, after which 2 drops of acetic acid were added to quench the reaction, and the polymer was precipitated into a 1:1 mixture of hexanes and diethylether. The polymer was redissolved in a minimal amount of acetone, and precipitated again in diethyl ether, and dried in vacuo. GPC was used to verify that the molecular weight and dispersity of polymers meet our QC parameters.
The reaction mixture was then sparged with nitrogen for 10 min.
Next, a degassed solution (0.1 ml) of CuBr (3.7 mg/ml) and THPTA (16 mg/ml) was added. The reaction mixture was further sparged with nitrogen gas for 10 min. The reaction mixture was incubated for 16 h at room temperature and precipitated into diethylether in a 50 ml centrifuge tube to recover the polymer. The polymer was then dissolved in ethyl acetate and precipitated into diethyl ether, followed by collection and drying in vacuo. GPC was used to verify that the molecular weight was not altered by conjugation.

| General PNP hydrogel preparation
The 2:10 PNP hydrogel formulation contained 2 wt% HPMC-C 12 and 10 wt% PEG-PLA NPs in PBS. These gels were made by mixing a 2:3:1 weight ratio of 6 wt% HPMC-C 12 polymer solution, 20 wt% NP solution, and PBS. For TLR7/8a-NP gels, the PEG-PLA NPs were made up of a mixture of TLR7/8a conjugated NP and non-conjugated NP based on the desired dose of adjuvant. The solutions were mixed with an elbow mixer and loaded into a syringe. from Sigma product information). Gels were placed onto glass slides and imaged using a confocal LSM780 microscope. Samples were imaged using low intensity lasers to collect an initial level of fluorescence. Then a high intensity laser was focused on a region of interest (ROI) with a 25 μm diameter for 10 s in order to bleach a circular area.

| FRAP analysis
Fluorescence data was then recorded for 4 min to create an exponential fluorescence recovery curve. Samples were taken from different regions of each gel (n = 2-5). The diffusion coefficient was calculated according to the following equation 27 : where the constant γ D = τ 1/2 /τ D , with τ 1/2 being the half-time of the recovery, τ D the characteristic diffusion time, both yielded by the ZEN software, and ω the radius of the bleached ROI (12.5 μm).
The diffusivity of the HA antigen in PBS was calculated using the Stokes-Einstein Law Equation for diffusion 28 where k B is Boltzmann's constant, T is temperature in Kelvin, η is solvent viscosity, and R is solute hydrodynamic radius: where the R of the HA antigen was taken to be 60 Å 29 and the η for PBS was taken to be 0.8872 mPa.s at 25 C. 30

| Statistical analysis
Comparisons between two groups were conducted by a two-tailed Student's t test. One-way ANOVA test with a Tukey's multiple comparisons test was used for comparison across multiple groups. Statistical analysis was run using GraphPad Prism 7.04 (GraphPad Software).
Statistical significance was considered as p < 0.05.

| RESULTS
Our previous work has shown that PNP hydrogels can be loaded with vaccine components for injection and provide extended co-delivery of physicochemically disctinct subunit vaccine cargo comprising ovalbumin and poly(I:C). These hydrogels are formed rapidly by mixing aqueous solutions of dodecyl-modified hydroxypropylmethylcellulose (HPMC-C 12 ) with biodegradable polymeric NPs composed of poly(ethylene glycol)-b-poly(lactic acid) (Figure 1(a)). 14,16,[31][32][33][34][35] The HPMC-C 12 polymer adsorbs onto the PEG-PLA NPs, forming multivalent and dynamic non-covalent interactions that constitute the physical cross-links of the hydrogel structure (Figure 1(a)). 36 The simplicity of the PNP hydrogel synthesis via simple mixing, performed here with an elbow mixer or spatula, allows for ease of production and potential for large scale fabrication of PNP hydrogels with consistent mechanical and structural properties. 17,37 This platform is also highly tunable in physical properties by changing the ratio of HPMC-C 12 to NP to an aqueous solution.
In this work we sought to augment the PNP hydrogel delivery platform for sustained co-delivery of the influenza antigen hemagglutinin (HA), the most common antigen in standard influenza subunit vaccines, with a potent TLR7/8a adjuvant that has previously been shown to elicit strong titers against HA and has demonstrated promise for clinical translation in influenza vaccination. 38  to the NPs which constitute a structural motif within the PNP hydrogels of our system, according to literature reports (Figure 1(b)). 24,26 The TLR7/8a molecule was conjugated to PEG-PLA using coppercatalyzed "click" chemistry of an alkyne-modified TLR7/8a with azide-terminated PEG-PLA (Supplementary Figure S1). The TLR7/8aconjugated PEG-PLA was then physically mixed with standard PEG-PLA at predetermined ratios and nanoprecipitated into water to create TLR7/8a-NPs with a controlled density of TLR7/8a molecules on the corona of the NPs. Two different populations of TLR7/8a-NPs were fabricated using either a mixture of 10 wt% TLR7/8a-conjugated PEG-PLA with 90 wt% standard PEG-PLA, or a mixture of 50 wt% of both TLR7/8a-conjugated PEG-PLA and standard PEG-PLA. The density of TLR7/8a molecules on the NPs is denoted as 10% or 50% based on the loading of TLR7/8a-PEG-PLA used in the TLR7/8a-NP synthesis.
We hypothesized that differences in the physical properties of the hydrogels would impact vaccine cargo diffusivity and lead to differences in the observed humoral immune responses to hydrogelbased immunization (Figure 1(c)). We modulated gel composition by changing the ratio of HPMC-C 12 to NP to an aqueous buffer solution during mixing (Figure 1  We first examined the impact of PNP hydrogel formulation on antibody responses (Figure 6(b)). Each of the four different PNP hydrogel formulations described above were loaded with HA and TLR7/8a-NPs with a valency of 10% and a 20 μg TLR7/8a total dose.
We reported previously that the strongest gel formulation, 2:10, persists in the subcutaneous space for roughly 4 weeks while the weakest gel, 1:5, persists for only about 2 weeks. 14 Coupled with the diffusivity data described above, the stiffer 2:10 gels were expected to provide the longest duration of vaccine release. Evaluation of total IgG endpoint titers over time indicated that all hydrogels performed better than the Alum control ( Figure 6(b)(i)). The 2:10, 1:10, and 2:5 hydrogel formulations all performed similarly, and performed better than the 1:5 hydrogel formulation. Assessment of the area under the curve (AUC) of the IgG titers indicated that longer-term vaccine exposure from the stiffer PNP hydrogel formulations led to higher overall vaccine potency and durability ( Figure 6(b)(ii)). These observations are consistent with previous reports on slow vaccine delivery with PNP hydrogels, 14 but suggest that a threshold for exposure time-frame exists, beyond which slower vaccine delivery has a negligible added benefit. Based on these results, we chose to conduct all further studies using the 2:10 PNP hydrogel formulation.
We then sought to evaluate the effect of distribution of TLR7/8a throughout the gel at a single TLR7/8a dose (20 μg) using TLR7/8a-NPs with a 10% TLR7/8a valency and 50% TLR7/8a valency, mixed with standard PEG-PLA NPs to achieve the same TLR7/8a dose. This study was conducted to understand whether have the adjuvant molecules more densely gathered adjuvant on the 50% TLR7/8a-NPs or more spread out throughout the hydrogel on more particles in the   can provide useful design criteria for next-generation influenza vaccines provided potent, durable, and broad immunity.

| CONCLUSIONS
In conclusion, here we show that the self-assembled, injectable PNP

DATA AVAILABILITY STATEMENT
All data supporting the results in this study are available within the Article and its Supplementary Information. The broad range of raw datasets acquired and analyzed (or any subsets of it), which for reuse would require contextual metadata, are available from the corresponding author on reasonable request.