Gillie A Roth1, Olivia M Saouaf2, Anton A A Smith2, Emily C Gale3, Marcela Alcántara Hernández4,5, Juliana Idoyaga4,5,6,7, Eric A Appel1,2,6,7,8. 1. Department of Bioengineering, Stanford University, 443 Via Ortega, Stanford, California 94305, United States. 2. Department of Materials Science & Engineering, Stanford University, 496 Lomita Mall, Stanford, California 94305, United States. 3. Department of Biochemistry, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, United States. 4. Department of Microbiology & Immunology, Stanford University School of Medicine, 299 Campus Drive, Stanford, California 94305, United States. 5. Program in Immunology, Stanford University School of Medicine, 240 Pasteur Drive, Stanford, California 94305, United States. 6. Institute for Immunity, Transplantation & Infection, Stanford University School of Medicine, 240 Pasteur Drive, Stanford, California 94305, United States. 7. ChEM-H Institute, Stanford University, 290 Jane Stanford Way, Stanford, California 94305, United States. 8. Department of Pediatrics - Endocrinology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, United States.
Abstract
The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.
The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.
Entities:
Keywords:
biomaterials; drug delivery; hydrogels; immunoengineering; vaccines
Seasonal influenza
causes roughly 500,000 deaths annually worldwide,
and every “bad” flu year reminds us that current flu
vaccine technologies are outdated and inadequate. A virulent pandemic
such as the Spanish flu outbreak of 1918, which killed over 40 million
people worldwide, could kill hundreds of millions today from more
rapid transmission fueled by crowded cities and active global transportation.[1] Indeed, new influenza strains with the potential
to become pandemics continue to arise worldwide, including a recently
described swine influenza variant that comprises genetic mutations
from European and Asian birds.[2] An efficacious
vaccine would elicit a persistent antibody response and high-affinity
broadly neutralizing antibodies (bnAbs) against the structurally diverse
and rapidly mutating hemagglutinin (HA) viral envelope glycoprotein.[3,4] BnAbs are characterized by extensive somatic hypermutation (SHM)
occurring during affinity maturation in germinal centers (GCs) within
lymphoid organs.[5,6] Yet, vaccines promoting long-lived
GCs and prolonging the SHM process to generate high affinity bnAbs
remain elusive.[7−10] Three parameters are crucial to enhancing affinity maturation: (i)
sustained vaccine exposure to prolong affinity maturation, (ii) the
use of adjuvants such as toll-like receptor agonists (TLRa), and (iii)
precise codelivery of the subunit vaccine components.[9,11] While reports indicate that subunit vaccines comprising multiple
TLRa molecules elicit better immune memory and stronger antibody responses,[12−14] controlled encapsulation and release of physicochemically distinct
reagents has historically been challenging or impossible.[13,15,16] Furthermore, adjuvants alone
typically do not provide sufficient immunological driving forces for
promoting extensive affinity maturation, and reports indicate that
prolonged vaccine exposure can have a profound effect on the magnitude
and quality of the immune response.[17−24] Yet, there are few delivery platforms reported to date that are
able to achieve controlled codelivery of physicochemically distinct
antigens and TLRa molecules over prolonged time frames.[20,25] To address this challenge, we have developed a controlled release
technology for prolonged codelivery of an influenza subunit vaccine
comprising hemagglutinin and small molecule imidazoquinoline TLR7/8a,
which has been shown to be a potent driver of an efficacious influenza
vaccine response.[13]A common challenge
when delivering small molecule adjuvants is
their tendency to diffuse directly into circulation, often causing
systemic toxicity instead of targeted immune modulation in lymph tissues.[26] A popular solution for augmenting the pharmacokinetics
and biodistribution of small molecule adjuvants, especially those
which are fairly hydrophilic (i.e., logP < 2), is conjugation to
nanoparticles which increases the size compared to soluble adjuvants.
This increase in size improves passive diffusion to the lymph nodes
(LN), thereby decreasing systemic exposure and associated toxicities.[27] Furthermore, these nanoparticle formulations
are able to prolong the stability and enhance the potency of the adjuvants.[28] Nanoparticle conjugation or encapsulation of
adjuvants has been shown to improve the activation of antigen presenting
cells (APCs) such as macrophages and dendritic cells since nanoparticle
sizes can be tuned for optimal endocytosis.[13,26,29] Though nanoparticle conjugation is beneficial
for improving the ability of adjuvant molecules to reach the target
cells, they do not provide a solution for sustained retention of the
adjuvant for a more prolonged inflammatory response.Injectable
hydrogels and depot technologies provide an avenue for
achieving sustained release of vaccine cargo with minimally invasive
administration (i.e., through direct injection).[30−35] In previous work, we showed that polymer–nanoparticle (PNP)
hydrogels can be used as a prolonged delivery platform for vaccine
administration to enhance the magnitude and duration of GC responses,
improving the durability of antibody responses and enhancing antibody
affinity maturation.[19] PNP hydrogels are
held together by dynamic, multivalent noncovalent interactions between
two structural motifs, which include a cellulosic biopolymer and a
nanoparticle (NP), allowing for them to be easily injected through
standard needles while also creating a robust depot at their injection
site.[20,36−41] The simplicity and scalability of the fabrication process allows
for facile manufacturing, which is a necessary design constraint for
future translation.[42−44] These hydrogels are highly modular and can be adapted
to deliver a wide range of cargo. When the cargo is sufficiently large,
its diffusion will be restricted by the network of the hydrogel and
will diffuse at the same rate as the dynamic rearrangement of the
gel’s polymer network itself.[35,38,45]The unique cargo delivery features of the PNP
hydrogels allow for
creating dependable sustained codelivery of multicomponent vaccine
formulations comprising large cargo (e.g., those with a hydrodynamic
radius above ∼3 nm). Yet, when a molecule is smaller than the
polymer mesh (e.g., small molecule imidazoquinoline TLR7/8a molecules),
it is able to diffuse relatively unhindered by the polymer network.
In this work, we sought to engineer an approach to PNP hydrogel formulation
to create a platform for sustained codelivery of encapsulated antigens
with a potent and highly specific TLR7/8a molecule through conjugation
of the TLR7/8a to the NP structural motif within the PNP hydrogel
network. We show that the TLR7/8a activity is not affected by the
conjugation to the NP construct and PNP hydrogel mechanical properties
are unperturbed when formulated with TLR7/8a-conjugated NPs rather
than standard NPs. We confirm that NP-tethered TLR7/8a and a well-studied
influenza antigen hemagglutinin (HA) diffuse at similar rates within
these materials, allowing for sustained codelivery. Furthermore, we
demonstrate that sustained delivery of this influenza vaccine leads
to an antibody response with enhanced potency, durability, and breadth
compared to clinical adjuvant systems. Overall, this study demonstrates
the potential of the injectable PNP hydrogel platform to be adapted
toward sustained codelivery of diverse adjuvants and antigens of interest
to enhance humoral immune responses.
Materials
and Methods
Materials
HPMC (meets USP testing specifications), N,N-diisopropylethylamine (Hunig’s
base), diethyl ether, hexanes, N-methyl-2-pyrrolidone
(NMP), lactide (LA), dichloromethane (DCM), diazobicylcoundecene (DBU),
and 1-dodecylisocynate were purchased from Sigma-Aldrich and used
as received. Monomethoxy-PEG (5 kDa) purchased from Sigma-Aldrich
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.
Polymer Characterization
Once polymer
synthesis was
completed, 1H nuclear magnetic resonance (NMR) was performed
to determine number-average molecular weight (Mn) using an Inova 300.
All samples were dissolved in d-chloroform for characterization. Samples
were passed through two size exclusion chromatography columns (Resolve
Mixed Bed Low DVB, ID 7.8 mm, Mw range 200–600 000 g/mol,
Jordi Laboratories) in a mobile phase of N,N-dimethylformamide (DMF) with 0.1 M LiBr at 35 °C
and a flow rate of 1.0 mL/min (Dionex Ultimate 3000 pump, degasser,
and autosampler (Thermo Fisher Scientific)), and subsequently the
ASTRA software package (Wyatt Technology Corporation) was used to
obtain absolute molecular weight and polydispersity. A HELEOS II light
scattering detector (Wyatt Technology Corporation) operating at 659
nm and an Optilab T-rEX (Wyatt Technology Corporation) refractive
index detector operating at 658 nm were used for detection. Dn/dc
values for PEG and PLA, 0.0442 and 0.019, respectively, in the mobile
phase, were calculated usingafter having determined
the dn/dc values for
PEG and PEG-PLA polymers of known weight fractions (via 1H NMR spectroscopy) in the ASTRA software package by batch injection
of 3 samples of known concentrations into an Optilab T-rEX refractive
index detector.
Preparation of HPMC-C12
HPMC-C12 was prepared according to previously reported
procedures.[37] HPMC (1.0 g) was stirred
at 80 °C for 1
h until dissolved in NMP (40 mL) before removing from heat. In a separate
vessel, Hunig’s base (catalyst, ∼3 drops) and 1-dodecylisocynate
(105 mg, 0.5 mmol) were dissolved in NMP (5.0 mL). Once the HPMC solution
mixture had cooled to room temperature, the catalyst solution was
added dropwise, and this mixture was then stirred at room temperature
for 16 h. This solution was then poured into acetone for precipitation.
The precipitate was decanted, redissolved in water (∼2 wt %),
and dialyzed in dialysis tubing for 3–4 days. The polymer was
cryodesiccated and reconstituted to 60 mg/mL in sterile PBS.
Preparation
of PEG-PLA
PEG-PLA was prepared as previously
reported.[37] Monomethoxy-PEG (5 kDa; 0.25
g, 4.1 mmol) and DBU (15 μL, 0.1 mmol; 1.4 mol % relative to
LA) were dissolved in anhydrous dichloromethane (1.0 mL). LA (1.0
g, 6.9 mmol) in anhydrous DCM (3.0 mL) was mildly heated for dissolution.
The LA solution was added rapidly to the PEG/DBU solution and stirred
for 10 min. This reaction mixture was quenched and precipitated by
addition to a 1:1 hexane and ethyl ether solution. The synthesized
PEG-PLA precipitate was collected and dried under vacuum. Gel permeation
chromatography (GPC) was used to verify that the molecular weight
and dispersity of polymers met our quality control (QC) parameters.
Preparation of TLR7/8a-PEG-PLA
TLR7/8a-PEG-PLA was
prepared according to a literature report,[29] and the protocols will be briefly described here. Azide-PEG-PLA
was prepared using N3-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 was added to quench the reaction,
and the polymer was precipitated into a 1:1 mixture of hexanes and
diethyl ether. 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.A 20 mL scintillation vial
was charged with the TLR 7/8 agonist alkyne (14 mg, 30 μmol),
and azido poly(ethylene oxide)-b-poly(d,l-lactide) (PEG5 kDa-PLA20 kDa, 0.5 g, 20 μmol) was dissolved in NMP (4.0 mL) and flushed
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. This reaction mixture
was then further flushed with nitrogen gas for 10 min. The reaction
mixture was stirred for 16 h at room temperature and added to diethyl
ether in a 50 mL centrifuge tube for precipitation to recover the
polymer. The polymer was next 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.
Conjugation was confirmed by 1H NMR spectroscopy and increased
UV absorption as indicated by GPC (DMF) eluagram.
General Preparation
of PEG-PLA NPs, TLR7/8a-NPs, and AF647-NPs
NPs were prepared
as previously reported.[37,46] A 1 mL solution of
PEG-PLA in DMSO (50 mg/mL) was added in a dropwise
fashion to 10 mL of water at room temperature stirred at 600 rpm.
NPs were characterized by dynamic light scattering (DLS) to find the
NP diameter and zeta potential (PEG-PLA NPs, 31 ± 3 nm, −28
± 7 mV; TLR7/8a-PEG-PLA NPs, 31 ± 3 nm, −10 ±
7 mV) (SI Table 1). AF647 NPs were prepared
using a combination of PEG-PLA (25 mg) and unconjugated azide-PEG-PLA
(25 mg) and then functionalized following purification by mixing azide-functional
NPs (500 μL, 20 wt %) with AF647-DBCO (5 μL, 1 mg/mL).
General PNP Hydrogel Preparation
The PNP hydrogel formulation
contained 2 wt % HPMC-C12 and 10 wt % PEG-PLA NPs in PBS.
These gels were made by mixing a 2:3:1 weight ratio of 6 wt % HPMC-C12 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 nonconjugated NP based on the desired dose of adjuvant. The
solutions were mixed with an elbow mixer and loaded into a syringe.
Vaccine Formulations
The influenza vaccine contained
a 2 μg dose of Influenza A H1N1 (A/Brisbane/59/2007) hemagglutinin
(HA)(Sino Biological) and an approximate TLR 7/8 agonist dose of 50
μg. For the PNP hydrogels, the vaccine cargo was added at the
appropriate concentration into the PBS component of the gel before
adding the polymer and NP solutions, as described above. For AddaVax
(InvivoGen; squalene based oil-in-water nanoemulsion similar to MF59,
which is a proprietary adjuvant produced by Novartis) and Alhydrogel
(Alum; InvivoGen) vaccines, the formulations were prepared according
to the manufacturer’s instructions with a 2 μg dose of
HA.
In Vitro RAW-Blue Reporter Assay
The
RAW-Blue reporter cell line (InvivoGen, raw-sp) was used to measure
TLR7/8 agonist activity. Cells were cultured at 37 °C with 5%
CO2 in Dulbecco’s modified Eagle’s medium
(DMEM; Thermo Fisher Scientific) supplemented with d-glucose
(4.5 g/L), l-glutamine (2 mM), penicillin (100 U/mL)/streptomycin
(100 μg), zeocin (100 μg/mL; Invi-vogen), and 10% heat
inactivated fetal bovine serum (Atlanta Biologicals). Soluble TLR7/8a
(R848) or TLR7/8a-NPs (20 μL) at a final concentration of 5
μg/mL was added to a 96-well tissue culture treated plate. Approximately
100,000 cells in 180 μL of media were added to each well. Cells
were cultured for 20 h at 37 °C in a CO2 incubator
before following manufacturer instructions for SEAP quantification
(absorbance at 655 nm).
Gel Rheological Characterization
Rheological characterization
was conducted with a TA Instruments Discovery HR-2 torque-controlled
rheometer fitted with a Peltier stage. All measurements were performed
using a serrated 20 mm plate geometry at 25 °C. Dynamic oscillatory
frequency sweep measurements were performed with a constant torque
(2 μN·m; σ = 1.27 Pa) from 0.1 rad/s to 100 rad/s.
Steady shear experiments were performed from 0.1 to 100 s–1. Yield stress values were found using stress ramp experiments.
FRAP Analysis
Hydrogels were made as stated above,
each with a unique fluorescent component: (i) free fluorescein, (ii)
AF647-NP, (iii) rhodamine-conjugated HPMC-C12, or (iv)
His-tagged hemagglutinin conjugated with HIS-Lite-Cy3 Bis NTA-Ni complex.
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. 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 as[47]where the constant
γD = τ1/2/τD, with
τ1/2 being the
half-time of the recovery, τD the characteristic
diffusion time, both yielded by the ZEN software, and ω the
radius of the bleached ROI (12.5 μm).
Animal Protocol
All animal procedures were performed
in accordance with National Institutes of Health guidelines, with
the approval of Stanford Administrative Panel on Laboratory Animal
Care.
Mice and Vaccination
C57BL/6 (B6) mice purchased from
Charles River were used for study of immune response and housed at
Stanford University. Female mice from 6 to 10 weeks of age at the
beginning of the experiment were used. The mice were shaved several
days before vaccine administration and received a subcutaneous injection
(100 μL administration volume) of gel or bolus vaccine on their
backs under brief isoflurane anesthesia. Mouse blood was collected
via tail vein bleeds for survival studies or through cardiac puncture
for terminal studies.
Antibody Concentration
Serum IgG
antibody titers for
the influenza vaccine were measure using an ELISA. Ni-coated plates
(Thermofisher) were coated with HA (Sino Biological) at 2.5 μg/mL
in PBS for 1 h at 25 °C and blocked with PBS containing 1% BSA
for 1 h at 25 °C. A standard curve was created by pooling serum
and completing serial dilutions (2×) before adding to the plate,
and serum samples were diluted 1:200 (Alum group) or 1:1,000 (Gel
and AddaVax groups) and added to plates. After 2 h at °C, goat-anti-mouse
IgG Fc-HRP (1:10,000, Invitrogen, A16084) was added for 1 h at 25
°C. Plates were developed with TMB substrate (TMB ELISA Substrate
(High Sensitivity), Abcam). The reaction was stopped with 1 M HCl.
The plates were analyzed with a Synergy H1 Microplate Reader (BioTek
Instruments) at 450 nm. Serum antibody titers were calculated from
a standard curve and represented as the dilution required to reach
the detection limit.Serum IgG1, IgG2b, and IgG2c antibody titers
against A/Brisbane/59/2007 HA and IgG titers against A/California/07/2009,
A/Michigan/45/2015, and A/Puerto Rico/8-WG/1934 HA were measure using
an end point ELISA. Ni-coated plates (Thermofisher) were coated with
HA (Sino Biological) at 2.5 μg/mL in PBS for 1 h at 25 °C
and blocked with PBS containing 1% BSA for 1 h at 25 °C. First,
serum was diluted 1:250 and then 4-fold serial dilutions were performed
up to 1:4,096,000 dilution. Titrations were added to plates and after
2 h at 25 °C, HRP-conjugated goat-anti-mouse IgG1 (Abcam, ab97240),
IgG2b (Chondrex, 3016), IgG2C (Abcam, ab97255), or IgG (Invitrogen,
A16084) was added at a 1:10,000 dilution for 1 h at 25 °C. Plates
were developed with TMB substrate (TMB ELISA Substrate (High Sensitivity),
Abcam). The reaction was stopped with 1 M HCl. The plates were analyzed
with a Synergy H1Microplate Reader (BioTek Instruments) at 450 nm.
End point titers were defined as the reciprocal of the highest serum
dilution that gave an optical density above 0.1.
Statistical
Analysis
All statistical methods are indicated
in the figure captions. Comparisons between two groups were conducted
by a two-tailed Student’s t test. A 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
In prior work, we have described the synthesis of injectable PNP
hydrogel materials that are highly efficient in loading vaccine components
for tunable codelivery of subunit vaccine components over prolonged
time frames.[20] These PNP hydrogels form
rapidly upon mixing of aqueous solutions of hydroxypropyl methylcellulose
derivatives (HPMC-C12) with biodegradable polymeric NPs
composed of poly(ethylene glycol)-b-poly(lactic acid)
(PEG-PLA) (Figure a).[46] Following mixing with an elbow mixer
or a spatula, the two solutions form multivalent and dynamic noncovalent,
multivalent interactions between the PEG-PLA NPs and the hydrophobically
modified HPMC polymer which creates the physical cross-links that
form to the hydrogel structure (Figure b). This facile synthesis allows the creation of hydrogel
formulations with a range of mechanical properties by simply changing
the ratio of HPMC-C12 to NP to an aqueous solution.[37] For this manuscript, we chose to use a 2 wt
% HPMC-C12 + 10 wt % NP formulation due to the improved
efficacy seen with this formulation in the delivery of OVA-based subunit
vaccines.[20]
Figure 1
Fabrication of polymer–nanoparticle
(PNP) hydrogels comprising
TLR7/8a-functional nanoparticles. (a, b) PNP hydrogels are formed
when (i) poly(ethylene glycol)-b-poly(lactic acid)
(PEG-PLA) nanoparticles (NPs) or TLR7/8a-conjugated PEG-PLA NPs are
combined with (ii) dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12). Multivalent and dynamic noncovalent interactions between
the polymer and NPs constitute physical cross-links that form the
hydrogel structure. Vaccine cargo can be added to the aqueous NP solution
before mixing, which yields complete encapsulation into the fabricated
hydrogels. (b) (iii) A homogeneous gel is easily achieved using an
elbow mixer or a spatula. (c) To ensure small molecule cargo such
as TLR7/8a achieves sustained delivery, it can be chemically conjugated
to the PEG-PLA NP structural motif of the hydrogels. (d) NHS coupling
of alykyne functionality to TLR7/8a (I) followed by copper-catalyzed
“click” coupling to azide-terminated PEG-PLA (II) yields PEG-PLA with the TLR7/8a (purple) presenting on
the hydrophilic PEG (blue) terminus of the block copolymer (III). This polymer is then nanoprecipitated into water to
form TLR7/8a-functional NPs.
Fabrication of polymer–nanoparticle
(PNP) hydrogels comprising
TLR7/8a-functional nanoparticles. (a, b) PNP hydrogels are formed
when (i) poly(ethylene glycol)-b-poly(lactic acid)
(PEG-PLA) nanoparticles (NPs) or TLR7/8a-conjugated PEG-PLA NPs are
combined with (ii) dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12). Multivalent and dynamic noncovalent interactions between
the polymer and NPs constitute physical cross-links that form the
hydrogel structure. Vaccine cargo can be added to the aqueous NP solution
before mixing, which yields complete encapsulation into the fabricated
hydrogels. (b) (iii) A homogeneous gel is easily achieved using an
elbow mixer or a spatula. (c) To ensure small molecule cargo such
as TLR7/8a achieves sustained delivery, it can be chemically conjugated
to the PEG-PLA NP structural motif of the hydrogels. (d) NHS coupling
of alykyne functionality to TLR7/8a (I) followed by copper-catalyzed
“click” coupling to azide-terminated PEG-PLA (II) yields PEG-PLA with the TLR7/8a (purple) presenting on
the hydrophilic PEG (blue) terminus of the block copolymer (III). This polymer is then nanoprecipitated into water to
form TLR7/8a-functional NPs.To apply this platform toward the influenza antigen hemagglutinin
(HA), which is the most commonly used antigen in influenza subunit
vaccines, we further adapted the hydrogel to ensure sustained codelivery
of the subunit components. In these studies, we used a TLR7/8a adjuvant
because TLR7/8a has been previously shown to elicit strong titers
against HA and has demonstrated promise for clinical translation.[13] To ensure sustained coadministration of HA and
the TLR7/8a, which is a small molecule (314 Da), we tethered the TLR
7/8a to the PEG-PLA NPs that form the PNP hydrogel network together
with HPMC-C12 (Figure c,d). To synthesize the TLR7/8a conjugated PEG-PLA,
alkyne modified TLR7/a was coupled to azide terminated PEG-PLA. NMR
was used to confirm conjugation (SI Figure 1). To generate TLR7/8a-NPs, the TLR 7/8a conjugated PEG-PLA polymers
were mixed in a 1:1 ratio with unconjugated PEG-PLA and nanoprecipitated
into water.It was important to confirm that the TLR7/8a conjugation
did not
affect the NP properties or the immunogenicity of the adjuvant, so
the TLR7/8a-NP were characterized with multiple assays. The TLR7/8a-NP
and plain NPs were characterized using dynamic light scattering (DLS)
and were both found to have hydrodynamic diameters (DH)
of 31 ± 3 nm s.d. (Figure a, b, SI Table 1), showing that
the adjuvant conjugation did not alter the NP diameter. To ensure
that TLR7/8a-NPs were still able to activate innate immune cells,
RAW-Blue macrophage cells were incubated for 20 h with either TLR7/8a-NP
or soluble TLR7/8a (R848) and their NF-kB and AP-1 activation was
measured. We saw similar levels of activation for soluble and conjugated
TLR7/8a-NP with absorbance values of 0.23 and 0.24, respectively (Figure c), verified with
a titrated range of adjuvant concentrations[28] (SI Figure 2). TLR7/8a activity depends
on multiple factors, including cell uptake (which can be enhanced
by nanoparticles), concentration, and potency (which can be affected
by valency).[27] These assays demonstrated
that we were able to synthesize stable TLR7/8a-NPs capable of potent
TLR7/8 activation.
Figure 2
Nanoparticle and gel characterization. (a) Characteristic
dynamic
light scattering (DLS) curves for PEG-PLA NPs (teal) and TLR7/8a-NPs
(dark blue). (b) Hydrodynamic diameters for PEG-PLA NPs and TLR7/8a-NPs
for four independent experiments measured with DLS (n = 4). (c) Activation of RAW-Blue macrophage cells after a 20-h incubation
with soluble TLR7/8a, TLR7/8a-NPs, or unconjugated NPs at 5 μg/well
of the adjuvant. Activation was determined using QUANTI-Blue (n = 3). (d) Frequency-dependent (σ = 1.8 Pa, 25 °C)
oscillatory shear rheology and (e) steady shear rheology of the PNP
gel with PEG-PLA NPs (dark blue) or TLR7/8a-NPs (teal). (f) Yield
stress values from stress ramp measurements (n =
3). All error bars are mean ± s.d. and P values were determined
by a two-tailed t test (b, f) or one-way ANOVA with
Tukey’s post hoc test (c).
Nanoparticle and gel characterization. (a) Characteristic
dynamic
light scattering (DLS) curves for PEG-PLA NPs (teal) and TLR7/8a-NPs
(dark blue). (b) Hydrodynamic diameters for PEG-PLA NPs and TLR7/8a-NPs
for four independent experiments measured with DLS (n = 4). (c) Activation of RAW-Blue macrophage cells after a 20-h incubation
with soluble TLR7/8a, TLR7/8a-NPs, or unconjugated NPs at 5 μg/well
of the adjuvant. Activation was determined using QUANTI-Blue (n = 3). (d) Frequency-dependent (σ = 1.8 Pa, 25 °C)
oscillatory shear rheology and (e) steady shear rheology of the PNP
gel with PEG-PLA NPs (dark blue) or TLR7/8a-NPs (teal). (f) Yield
stress values from stress ramp measurements (n =
3). All error bars are mean ± s.d. and P values were determined
by a two-tailed t test (b, f) or one-way ANOVA with
Tukey’s post hoc test (c).To ensure that the conjugation of TLR7/8a to the NPs did not influence
the mechanical properties of the PNP hydrogels, rheological characterization
comparing hydrogels formulated with TLR7/8a-NPs and standard PEG-PLA
NPs was performed. Frequency-dependent oscillatory shear experiments,
performed in the linear viscoelastic regime, demonstrated that TLR7/8a
conjugation to the NP does not influence the PNP hydrogels’
frequency-dependent rheology (Figure d). At a representative angular frequency (ω
= 10 rad/s), the standard PNP gel and TLR7/8a-NP gel exhibited storage
moduli (G′) of 350 and 210 Pa, respectively.
The angular frequency sweep rheological data also showed that both
unadjuvanted and adjuvanted gels exhibited solid-like properties across
the full range of frequencies tested, with the G′
remaining above the loss modulus (G′′)
at all frequencies evaluated (Figure d). To compare the gels’ abilities to shear-thin
and behave similarly in injection conditions, a shear rate sweep was
performed. The viscosity of both PNP hydrogels decreased around 3
orders of magnitude as shear rates increased and exhibited similar
shear-rate dependent viscosities (Figure e). A stress ramp showed the yield stress
of these materials to be approximately 300 and 250 Pa for the standard
PNP gel and TLR7/8a-NP gels, respectively (Figure f). These yield stress values demonstrate
each gels’ ability to remain solid-like under low stresses
(i.e., pre- and postinjection), thereby preserving a robust gel structure
postinjection and inhibiting dissipation via flow from the injection
site. This feature is critical for facilitating the creation of a
local inflammatory niche.[20,48,49]To characterize the diffusion rates of vaccine components
within
the gel, fluorescence recovery after photobleaching (FRAP) measurements
was performed (Figure a). In these experiments, a fluorescent moiety was conjugated to
NPs, HA, or HPMC-C12 and these components were formed into
a gel (free fluorescein served as a proxy for the similarly sized
small molecule cargo TLR7/8a). A defined circular region of the gel
was bleached via exposure to intense light. Fluorescence recovery
was measured as particles from outside the region diffused in, generating
a curve from which the diffusivity of the gel components and entrapped
cargo was calculated (Figure b). Small molecule cargo such as free fluorescein was shown
to diffuse faster than any other component in the gel, indicating
that untethered TLR7/8a is smaller than the gel’s mesh size,
diffusing rapidly out of the gel and thereby not capable of sustained
codelivery with HA antigen (Figure c, SI Table 2). When compared
to the diffusivity of the polymer matrix, measured as the diffusivity
of HPMC-C12, both NPs and HA had ratios close to 1, demonstrating
that both HA and NP-tethered TLR7/8a are “caught” within
the hydrogel mesh and diffuse with the network as intermolecular interactions
break and reform (Figure d). Finally, a comparison of NP-tethered and untethered moieties
to the diffusion of HA indicates that NP-tethered TLR7/8a achieves
codelivery with the antigen as their ratio is close to 1, while untethered
adjuvant diffuses much more quickly than the antigen (Figure e). Assuming a spherical 100
μL gel (the typical administration volume for our mouse studies, vide infra), Brownian motion approximates the time frame
of HA (D = 1.1 ± 0.18 μm2/s) and TLR7/8a-NP
(D = 1.0 ± 0.48 μm2/s) release from the gel
to be 16 and 14.5 days, respectively, indicating that our delivery
platform enables prolonged codelivery of antigen and adjuvant over
the course of 2–3 weeks (SI-Cargo Release Time).
Figure 3
Diffusivity
of vaccine and gel components. (a) FRAP measurements
are taken of dye-conjugated components of the gel or entrapped dye-conjugated
cargo by photobleaching a circular region and measuring the recovery
of fluorescence as dye-conjugated species diffuse back into the photobleached
region. (b) Representative graph of fluorescence recovery data for
the small molecule fluorescein in a PNP gel. (c) Diffusivity calculated
from FRAP measurements in a PNP gel (n = 2–5)
for each fluorescently labeled gel component and vaccine component:
model small molecule cargo (untethered fluorescein representing untethered
TLR7/8a), NP (NP-tethered AF647 representing TLR7/8a-NP), HA (Cy3-labeled
HA), and polymer matrix (rhodamine-conjugated HPMC-C12).
(d) Component diffusivities normalized by Dgel (the polymer
matrix diffusivity) show NPs and HA are caught in the hydrogel network.
Dotted line is at D/Dgel = 1. (e) Measured diffusivities
demonstrate that molecules attached to NPs (NP) diffuse at a similar
rate to HA, allowing for sustained codelivery, while untethered small
molecule cargo quickly diffuses out of the gel. Dotted line is at
D/DHA = 1.
Diffusivity
of vaccine and gel components. (a) FRAP measurements
are taken of dye-conjugated components of the gel or entrapped dye-conjugated
cargo by photobleaching a circular region and measuring the recovery
of fluorescence as dye-conjugated species diffuse back into the photobleached
region. (b) Representative graph of fluorescence recovery data for
the small molecule fluorescein in a PNP gel. (c) Diffusivity calculated
from FRAP measurements in a PNP gel (n = 2–5)
for each fluorescently labeled gel component and vaccine component:
model small molecule cargo (untethered fluorescein representing untethered
TLR7/8a), NP (NP-tethered AF647 representing TLR7/8a-NP), HA (Cy3-labeled
HA), and polymer matrix (rhodamine-conjugated HPMC-C12).
(d) Component diffusivities normalized by Dgel (the polymer
matrix diffusivity) show NPs and HA are caught in the hydrogel network.
Dotted line is at D/Dgel = 1. (e) Measured diffusivities
demonstrate that molecules attached to NPs (NP) diffuse at a similar
rate to HA, allowing for sustained codelivery, while untethered small
molecule cargo quickly diffuses out of the gel. Dotted line is at
D/DHA = 1.Next, to interrogate
how sustained codelivery of antigen and adjuvant
influences humoral immunity toward the HA antigen from A/Brisbane/59/2007
(H1N1), we quantified the HA-specific IgG antibody titers after single
subcutaneous administration of HA in the TLR7/8a-NP gels compared
with unconjugated TLR7/8a (R848) in a standard gel (TLR7/8a-Sol Gel).
We also compared these formulations to delivering HA as a bolus with
clinically relevant adjuvants AddaVax, which has a similar formulation
to MF59, the most potent influenza vaccine adjuvant used clinically,
and Alum, an aluminum hydroxide adjuvant which has been used previously
in clinical influenza vaccine formulations[50,51] (Figure a,b). Subcutaneous
delivery, used to allow distinct gel depot formation, is a route of
administration for multiple clinical vaccines.[52] We showed that 56 days after administration, the TLR7/8a-NP
gels led to 6-fold, 19-fold, and 126-fold higher antibody titers compared
to AddaVax, TLR7/8a-Sol gel, and Alum, respectively. Additionally,
mice receiving the gel-based vaccine maintained antibody titers above
the AddaVax group’s peak titer for over 140 days (SI Figure 3). The increased area under the curve
(AUC) of the titers over time demonstrates that the TLR7/8a-NP gel
vaccine formulation led to more potent and durable antigen-specific
humoral immune responses compared to TLR7/8a-Sol gel, AddaVax, and
Alum (Figure c). We
saw that the TLR7/8a-NP gels led to higher anti-HA IgG titers than
the TLR7/8a-Sol gel and bolus administration of TLR7/8a-NPs or soluble
TLR7/8a (R848) (Figure d,c, SI Figure 4). These studies demonstrated
that having prolonged codelivery of both the antigen and adjuvant
is critical for the benefits we measured. Further characterization
of the IgG subclasses 56 days after single administration showed that
TLR7/8a-NP gel delivery led to a significant increase in IgG1, IgG2b,
and IgG2c antibodies compared to both adjuvant controls (Figure d–f). Notably,
we saw an 84-fold increase in IgG2c titers for the TLR7/8a-NP gel-based
vaccine compared to AddaVax, which is the most important IgG subclass
in C57BL/6 mice for fighting viral infections.[53]
Figure 4
Humoral response to influenza hemagglutinin subunit vaccine. (a)
A 2 μg dose of hemagglutinin (HA) was administered subcutaneously
in either a PNP gel formulated with 10% TLR7/8a-conjugated nanoparticles
(TLR7/8a-NP Gel) and 2% HPMC-C12, 10% unconjugated nanoparticles
and soluble TLR7/8a (TLR7/8a-Sol Gel) and 2% HPMC-C12,
a bolus of AddaVax (formulated like MF59, the most potent adjuvant
used clinically for influenza), or Alum. (b) Serum anti-HA IgG titers
from day 14 to day 56 after single injection of HA delivered in TLR7/8a-NP
gel, TLR7/8a-Sol gel, an AddaVax bolus, or Alum bolus. P values for
TLR7/8a-NP gel compared to Alum (bottom, dark blue), TLR7/8a-Sol gel
(middle, light blue), or AddaVax (top, orange) are shown (n = 4 to 5). (c) Area under the curve (AUC) of anti-HA IgG
titers (n = 4 to 5) from (b). Serum anti-HA IgG1
(d), IgG2b (e), and IgG2c (f) titers from day 56 after single injection
of HA delivered in the TLR7/8a-NP gel, TLR7/8a-Sol gel, AddaVax bolus,
or Alum bolus. All error bars are mean ± s.d., P values determined
by two-way ANOVA with Tukey’s post hoc test (b) or one-way
ANOVA with Tukey’s post hoc test (c–f).
Humoral response to influenza hemagglutinin subunit vaccine. (a)
A 2 μg dose of hemagglutinin (HA) was administered subcutaneously
in either a PNP gel formulated with 10% TLR7/8a-conjugated nanoparticles
(TLR7/8a-NP Gel) and 2% HPMC-C12, 10% unconjugated nanoparticles
and soluble TLR7/8a (TLR7/8a-Sol Gel) and 2% HPMC-C12,
a bolus of AddaVax (formulated like MF59, the most potent adjuvant
used clinically for influenza), or Alum. (b) Serum anti-HA IgG titers
from day 14 to day 56 after single injection of HA delivered in TLR7/8a-NP
gel, TLR7/8a-Sol gel, an AddaVax bolus, or Alum bolus. P values for
TLR7/8a-NP gel compared to Alum (bottom, dark blue), TLR7/8a-Sol gel
(middle, light blue), or AddaVax (top, orange) are shown (n = 4 to 5). (c) Area under the curve (AUC) of anti-HA IgG
titers (n = 4 to 5) from (b). Serum anti-HA IgG1
(d), IgG2b (e), and IgG2c (f) titers from day 56 after single injection
of HA delivered in the TLR7/8a-NP gel, TLR7/8a-Sol gel, AddaVax bolus,
or Alum bolus. All error bars are mean ± s.d., P values determined
by two-way ANOVA with Tukey’s post hoc test (b) or one-way
ANOVA with Tukey’s post hoc test (c–f).To evaluate cross reactivity of serum to HA variants not
included
in the vaccine formulation, total IgG titers against HA from “future”
influenza variants A/California/07/2009(H1N1) (Cal09) and A/Michigan/45/2015(H1N1)
(Mich15), as well as A/PuertoRico/8/1934(H1N1) (PR8) (SI Figure 5), were quantified (Figure ). Serum from animals immunized
with A/Brisbane/59/2007(H1N1) (Bris07) HA in TLR7/8a-NP gels exhibited
a 16-fold, 64-fold, and 3.4-fold increase in titers against Cal09,
Mich15, and PR8 HA variants compared to delivery with AddaVax adjuvant,
respectively, and a 256-fold, 64-fold, and 16-fold increase in titers
against Cal09, Mich15, and PR8 HA variants compared to delivery with
Alum adjuvant, respectively (Figure c–e). The breadth can also be visualized by
plotting the heterologous titers (Cal09, Mich15, PR8) against the
homologous titer (Bris07) (Figure f–g). These plots further demonstrate the increase
in vaccine potency across multiple “future” HA variants
after single administration of Bris07 HA in the TLR7/8a-NP gel. Remarkably
the difference in titer between the TLR7/8a-NP gel and the TLR7/8a-Sol
gel was much higher with these heterologous HA variants compared to
the homologous HA, demonstrating the influence of prolonged codelivery
of antigen and adjuvant on improving the breadth of the antibody response.
Taken together, these results indicate that prolonged codelivery of
HA and a TLR7/8a in our hydrogel platform significantly increases
the breadth of the antibody response compared to the most potent clinical
adjuvants.
Figure 5
Increased breadth of antibodies toward future influenza strains.
(a) Phylogenetic tree of A/Michigan/45/2015(H1N1) (Mich15), A/California/07/2009(H1N1)
(Cal09), A/Brisbane/59/2007(H1N1) (Bris07), and A/Puerto Rico/8-WG/1934(H1N1)
(PR8) influenza antigens based on HA DNA sequence where the branch
lengths represent modifications per site. The tree was created using
the ‘Generate Phylogenetic Tree’ tool on the NIAID Influenza
Research Database (IRD) through the Web site at http://www.fludb.org. (b) Experimental
schematic showing how ELISAs can be used to measure the breadth of
anti-HA responses. Anti-HA titers for Cal09 (c), Mich15 (d), and PR8
(e) for serum from day 56 after single injection of Bris07 HA antigen
delivered in the TLR7/8a-NP gel, TLR7/8a-Sol gel, or as a bolus with
AddaVax or Alum. Cal09 (f), Mich15 (g), and PR8 (h) titers plotted
against Bis07 titers demonstrated that the TLR7/8a-NP gels led to
the most potent antibody response across multiple HA variants. All
error bars are mean ± s.d., P values determined by one-way ANOVA
with a Tukey’s post hoc test.
Increased breadth of antibodies toward future influenza strains.
(a) Phylogenetic tree of A/Michigan/45/2015(H1N1) (Mich15), A/California/07/2009(H1N1)
(Cal09), A/Brisbane/59/2007(H1N1) (Bris07), and A/Puerto Rico/8-WG/1934(H1N1)
(PR8) influenza antigens based on HA DNA sequence where the branch
lengths represent modifications per site. The tree was created using
the ‘Generate Phylogenetic Tree’ tool on the NIAID Influenza
Research Database (IRD) through the Web site at http://www.fludb.org. (b) Experimental
schematic showing how ELISAs can be used to measure the breadth of
anti-HA responses. Anti-HA titers for Cal09 (c), Mich15 (d), and PR8
(e) for serum from day 56 after single injection of Bris07 HA antigen
delivered in the TLR7/8a-NP gel, TLR7/8a-Sol gel, or as a bolus with
AddaVax or Alum. Cal09 (f), Mich15 (g), and PR8 (h) titers plotted
against Bis07 titers demonstrated that the TLR7/8a-NP gels led to
the most potent antibody response across multiple HA variants. All
error bars are mean ± s.d., P values determined by one-way ANOVA
with a Tukey’s post hoc test.
Discussion
The results described in this study provide multiple insights into
how to use material strategies to better control the delivery of adjuvants
and antigens, specifically TLR7/8 agonists and HA, for improving influenza
vaccination. We demonstrate that the conjugation of TLR7/8a to PEG-PLA
NPs is able to increase the hydrodynamic diameter of the small molecule,
while maintaining its bioactivity. For our application this allowed
the TLR7/8a to have slower diffusion in the PNP gel and therefore
have matched delivery kinetics to the HA as shown by FRAP experiments.
The approach of tethering adjuvants to NPs can be more broadly applied
to prolonged delivery hydrogels, as the increase hydrodynamic radius
will enable prolonged release across many different materials platforms.We see that the slow codelivery of TLR7/8a with antigen is critical
in achieving improved antibody titers and breadth, as the soluble
TLR7/8a (R848) formulation of the vaccine did not match the performance
of the TLR7/8a-NP gel. Furthermore, bolus administration of the TLR7/8a-NP
on their own with the HA antigen did not lead to improvements in antibody
responses. From these results we can conclude that sustained codelivery
of both the antigen and adjuvant is beneficial to initiating a broad
anti-HA antibody response. We have previously shown that sustained
delivery of the OVA and Poly(I:C) model vaccine led to an inflammatory
niche and prolonged GCs after a single administration.[19] We expect that the TLR7/8a-NP gel delivering
HA has the same effect on the vaccine response, whereby prolonging
the exposure to both antigen and adjuvant within the PNP hydrogel
creates a local environment where TLR7/8a and HA are able to have
extended interactions with innate immune cells due to their similar
diffusion release profiles. These cells secrete critical cytokine
and chemokines to attract more cells toward the site of injection
and enhance antigen processing and presentation. In the GCs, the extended
vaccine release allows for prolonged GCs and more cycles of affinity
maturation. This guides the immune response toward the creation of
higher affinity antibodies and could lead to the increased breadth
that we demonstrated.An important aspect of influenza vaccine
development is to find
strategies to initiate potent and durable antibody responses against
“future” strains of influenza that are not incorporated
in the vaccine itself. To compare the impact of slow Bris07-based
vaccine delivery in the TLR7/8a-NP gel compared to AddaVax, which
is the most potent clinical adjuvant system currently used, we can
characterize breadth as a ratio of the titer against a heterologous
HA to the titer against the homologous HA (i.e., titerCal09 or Mich09/titerBris07). Such an analysis shows that TLR7/8a-NP
gels exhibited 2.3-fold and 3.9-fold greater breadth of responses
than AddaVax for Cal09 and Mich15, respectively. These observations
corroborate previous work evaluating the impact of slow HIV vaccine
delivery on the breadth of responses[21] and
suggests that prolonged delivery technologies are a powerful tool
for achieving more potent, durable, and broad vaccine responses and
may play an important role in developing a truly universal flu vaccine.
Conclusions
In conclusion, sustained delivery of subunit influenza vaccines
can be achieved using PNP hydrogels to increase the potency, durability,
and breadth of the humoral immune response. We have shown that the
PNP hydrogel delivery platform can be adapted through conjugation
of small molecule adjuvants to structural motifs within the hydrogels
in order to engineer a truly modular sustained delivery system to
prolong influenza subunit vaccine exposure. This facile and mild fabrication
of these materials by simple mixing can enable robust encapsulation
of essentially any antigen, while the precisely controlled delivery
characteristics can enable future studies aimed at elucidating the
mechanisms by which slow exposure kinetics improve vaccine responses.
When paired with sophisticated antigens, the PNP hydrogel delivery
system has the potential to form the basis of next-generation vaccines
against challenging pathogens such as influenza that continue to pose
enormous risks to public health worldwide.
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