Article pubs.acs.org/bc
Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant Joseph R. Francica,† Geoffrey M. Lynn,† Richard Laga,‡ M. Gordon Joyce,† Tracy J. Ruckwardt,† Kaitlyn M. Morabito,† Man Chen,† Rajoshi Chaudhuri,§ Baoshan Zhang,† Mallika Sastry,† Aliaksandr Druz,† Kiyoon Ko,† Misook Choe,† Michal Pechar,‡ Ivelin S. Georgiev,† Lisa A. Kueltzo,§ Leonard W. Seymour,∥ John R. Mascola,† Peter D. Kwong,† Barney S. Graham,† and Robert A. Seder*,† †
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States ‡ Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague, Czech Republic § Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Gaithersburg, Maryland 20878, United States ∥ Department of Oncology, University of Oxford, Oxford OX3 7DQ, U.K. S Supporting Information *
ABSTRACT: Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Tolllike receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusionspecific, TH1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.
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INTRODUCTION
need to optimize both the antigen and the adjuvant to maximize adaptive immune responses. Until recently, aluminum salts (“alum”) or oil-and-water emulsions were the only approved adjuvants available to enhance antibody responses to protein subunit vaccines. More recently, Toll-like receptor agonists (TLRa) have been used as vaccine adjuvants.11 Monophosphoryl lipid A (MPL), a TLR4a, has been approved for human use against human papillomavirus (HPV) when administered with alum and shows increased antibody responses compared to alum alone.12 Similarly, subunit vaccines containing MPL enhance immunogenicity and protection against malaria13 and hepatitis B.14 The safe and successful use of MPL has provided the necessary proof of concept for advancing the development of other TLRa that may
Structural vaccinology has been used to improve the design of protein immunogens for vaccination against a number of infectious diseases.1,2 Integral to this process is the presentation of conserved neutralizing epitopes, shielding of non-neutralizing sites, and the prevention of conformational changes that can present immunodominant non-neutralizing epitopes. This immunofocusing reduces “off-target” immune responses and may enhance responses to protective neutralizing epitopes.3 Structure-based vaccine design has been applied to the HIV-1 envelope (Env),4−6 influenza hemagglutinin (HA),7 Epstein− Barr virus gp350,8 and respiratory syncytial virus fusion (RSV F)3 proteins. However, such subunit vaccines alone are often less immunogenic than intact or live-attenuated pathogens9 and so must be formulated with an exogenous adjuvant to improve the magnitude and quality of humoral and cellular immunity. Even multivalently arrayed subunit vaccines that cross-link B cell receptors are often further improved by adjuvants to increase the magnitude of antibody titers,7,8,10 highlighting the © 2016 American Chemical Society
Received: Revised: Accepted: Published: 2372
July 8, 2016 August 29, 2016 September 1, 2016 September 1, 2016 DOI: 10.1021/acs.bioconjchem.6b00370 Bioconjugate Chem. 2016, 27, 2372−2385
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Figure 1. Primary amine coupling of TLR-7/8a molecules to RSV F and disruption of antigenicity. (a) The trimeric F glycoprotein is shown in surface representation with each protomer colored differently. Colored outlines indicate footprints of known sites of neutralization; primary amine containing residues are colored brown. (b) Distance of primary amine residues to known sites of neutralization on the F trimer; residues that are part of the epitope as well as adjacent residues (defined by a 5 Å cut-off) are highlighted in bold. The measured distance is from the primary amine atom to the closest atom of the antibody epitope using previously described antigenic sites (site ⌀: 62-69, 196-209; site I: 387-392; site II: 254-276; site IV: 427-436) and the AM14-F crystal structure (PDB ID: 4ZYP). (c)The TLR-7/8a, 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4amine (2BXy), was coupled to trimeric F protein (DS-Cav1) in a two-step reaction. First, F protein was functionalized with azide groups through primary amines using either a 20:1 (hi) or 10:1 (lo) molar ratio of NHS-PEG4-Azide to F protein. In the second step, DBCO-modified 2BXy was coupled to the azide-functionalized protein to generate an F-TLR-7/8a conjugate. (d-g) Trimeric F protein was conjugated with TLR-7/8a using primary amine chemistry at two molar ratios of agonist to protein: 10:1, “lo” and 20:1, “hi”. The binding to RSV F mAbs AM14 (d), D25 (e), MPE8 (f), and Motavizumab (g) to serial dilutions (646−162 nM) of unconjugated and conjugated protein was assayed by biolayer interferometry. Colored traces depict raw binding data at 646 nM; black traces indicate fitting data; vertical dotted lines indicate the transition from the association to dissociation phase. (h) Binding affinity constants were derived from global fitting of the kinetic data to each mAb at each temperature using a 1:1 Langmuir binding equation.
antigen-presenting cells (APC) has been shown to be beneficial for priming T cell immunity.25−30 We and others have shown that direct chemical conjugation of TLR-7/8a to immunogens is an effective co-delivery approach for inducing T cell immunity.31−33 However, the chemical modification of proteins to link TLR-7/8a may result in a loss of antigenicity31 and aggregation32 of the immunogen, which limits this approach for antibody-based vaccines. To address these challenges, we recently showed that synthetic, biocompatible polymer chains are an effective vehicle for improving the pharmacokinetic properties of TLR-7/8a and coupling them with protein immunogens to ensure codelivery.20 Accordingly, our previous work characterized thermoresponsive polymers (TRP) composed of single, amphiphilic, diblock co-polymer chains that are soluble at room temperature but can be induced to self-assemble into nanoparticles above a chemically programmable transition temperature. Coupling TLR-7/8a and protein immunogens to TRP nanoparticles improved APC uptake, leading to higher magnitude and persistence of protective cellular immune responses.20
further improve immunity and protection based on stimulation through alternative innate pathways. On the basis of unique patterns of TLR expression across different immune cell subsets, TLRa can be rationally selected to induce qualitatively distinct immune responses that would be most protective against a given pathogen.15 Accordingly, imidazoquinoline compounds have been identified that signal through TLR7 to directly activate plasmacytoid dendritic cells (DCs)16,17 and B cells18 and through TLR8 to stimulate monocytes and other DC subsets,16,19 leading to robust humoral and cellular immunity. However, small-molecule TLRa, e.g., TLR-7/8a, rapidly diffuse away from the site of injection, reducing their ability to prime immune cells and potentially causing systemic side effects.20,21 To focus activity in lymph nodes draining the sites of immunization while reducing systemic distribution, TLRa may be formulated with a delivery vehicle that prevents systemic dissemination22 and enhances uptake by immune cells. Insoluble aluminum salts,22 oil-inwater emulsions,23 and liposomal vesicles24 among others have been used for this purpose. Additionally, physically linking TLRa with protein immunogens to ensure their co-delivery to 2373
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Motavizumab (site II, prefusion and postfusion). Similarly, both apparent kon and koff rates of these mAbs were detrimentally affected by the TLR-7/8a conjugation (Figure 1h). These data show that direct coupling of TLR-7/8a using primary amine chemistry blocks antibody binding to critical sites of neutralization on the stabilized prefusion RSV F protein. Polymer Nanoscaffolds as a Platform for Co-delivery of TLR-7/8a and RSV F Protein. To develop an alternative to primary amine conjugation, synthetic polymers were used to link TLR-7/8a with RSV F protein while preserving its neutralizing epitope sites. We have previously shown that HIV Gag protein fused to a synthetic α-helical (“coil”) peptide can be site-specifically attached to polymer chains bearing both a complementary coil peptide and multiple TLR-7/8a to elicit strong T cell immunity.20 However, it has not been investigated how this approach influences the antigenicity of the protein or the generation of neutralizing antibody responses. Thus, we evaluated whether RSV F can be linked to polymer nanoparticles bearing TLR-7/8a via coil−coil protein interactions in a defined manner that does not interfere with the antigenic surface of the protein. As a modular platform for evaluating the site-specific attachment of RSV F protein to polymer carriers, thermoresponsive diblock co-polymers (TRP) composed of hydrophilic (HPMA) and hydrophobic (DEGMA) blocks were synthesized (Table S1 and Figure S2). Both the TLR-7/8a and synthetic αhelical peptides, abbreviated “ESE” (Figure 2), were attached to the hydrophilic block of the polymer to produce a thermoresponsive polymer-TLR-7/8a and -ESE coil peptide conjugate, referred to as TRP-7/8a-ESE. Placing the TLR-7/8a and ESE coil on the hydrophilic block avoids disruption of the temperature-dependent property of the hydrophobic block and provides greater accessibility to the solvent exposed portion of the polymer and, thus, to BCRs. The RSV F protein was recombinantly modified with an α-helical coil sequence, abbreviated as “KSK”, genetically fused to the C terminus via a 10 residue glycine-serine linker (F-KSK). This KSK coil is complementary to the ESE coil conjugated to the polymer. The ESE and KSK coils preferentially associate as antiparallel heterodimers via electrostatic and hydrophobic interactions, thus driving attachment of RSV F trimers to the TRP-7/8a-ESE conjugate20,46 (Figure 2). Finally, the DEGMA block endows these polymers with unique thermoresponsive properties, as previously described.20 Accordingly, above a transition temperature of approximately 30 °C, these polymer chains collapse into globules that exclude water molecules and associate with each other to form nanosized supramolecular assemblies (i.e., nanoparticles) (Figure 2). With this approach, nanoparticle formation is driven at body temperature, preferentially leaving the immunogen and adjuvant on or near the solvent-exposed surface of the particles. Characterization of Attachment of F-KSK to TRP-7/8ESE and Particle Formation. Dynamic light scattering (DLS) was used to characterize the hydrodynamic behavior of the TRP formulations used throughout this study (Figure S3a,b and Table S1). TRP-7/8a-ESE were found to undergo particle formation (RH from ∼7 to ∼14 nm) at a transition temperature of 33 °C. Similar results were obtained with polymers coupled to RSV F trimers, with an increase in particle size dependent on the ratio of RSV F-KSK to TRP-7/8a-ESE in the formulation (Figure S3c,d and Table S1). Importantly, the increase in size of the TRP-7/8-ESE particles with addition of F-KSK appeared to be dependent on the coil−coil interaction between “KSK”
The goal of this study was to investigate the utility of the TRP platform for co-delivering a structurally stabilized B cell immunogen with TLR-7/8a in a formulation that enhances immunogenicity while preserving critical neutralizing epitopes. To this end, the trimeric fusion (F) protein from RSV was selected as a model immunogen because the structural integrity of this immunogen is critical to the generation of protective neutralizing antibody responses.3,34−36 RSV virions have an array of F trimers on the viral membrane and a single-stranded RNA genome that can signal through TLR7 and TLR8.37 To mimic these properties of RSV, we investigated the effects of immunogen multivalency and co-delivery with TLR-7/8a on anti-RSV immunity. However, natural RSV infection does not confer protective immunity, in part because F trimers can exist on the virion surface in the postfusion conformation, acting as decoys to distract the immune response from neutralizing epitopes found only in the prefusion conformation.36 Thus, the ability of the TRP platform to preserve the F trimer prefusion conformation and focus antibody responses against specific neutralizing sites was assessed. Moreover, as a previous RSV vaccine that induced TH2-biased responses enhanced RSV illness in children,38−40 the ability of TLR-7/8a to promote TH1 skewed responses was also determined. Here, we provide the first evidence that a structurally stabilized immunogen can be site-selectively coupled with TLR-7/8a to polymer nanoparticles, preserving antigenicity and eliciting protective TH1 immunity.
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RESULTS Direct Conjugation of TLR-7/8a to RSV F Protein and Disruption of Antigenicity. Prior studies by us and others have shown that direct chemical conjugation of TLRa to protein antigens is an effective co-delivery approach for improving T cell immunogenicity.30,32,41,42 To advance this approach for inducing antibody responses, the stabilized prefusion RSV F trimer (DS-Cav1) was first analyzed for accessible primary amines to directly couple TLR-7/8a molecules (Figure 1a). In total, 32 residues on the surface of each protomer are available for amine coupling, composing ∼9% of the molecular surface area of the RSV F trimer (Figure 1b). However, 18 of 32 residues with primary amines are located within or adjacent to previously described RSV F sites of neutralization, suggesting that coupling TLR-7/8a to primary amines could physically block B cell receptor (BCR) recognition of neutralizing epitopes on RSV F trimers. To address this directly, primary amine chemistry was used to conjugate an imidazoquinoline-based TLR-7/8a (2BXy)43−45 to the surface of RSV F trimers using 10 or 20 mol equiv of the TLR-7/8a to F protein (F lo TLR-7/8a and F hi TLR-7/8a, respectively, Figures 1c and S1). The ability to generate protective antibody responses against RSV requires that neutralizing epitopes on the RSV F protein immunogen are accessible to binding by B cells. One means of assessing how a vaccine formulation influences the accessibility of sites of neutralization is to measure the binding of monoclonal antibodies (mAbs). Therefore, biolayer interferometry (BLI) was used to perform an antigenic assessment of the F-TLR-7/8a conjugates using mAbs against defined neutralizing epitope sites (Figure 1d−g). BLI demonstrated that direct coupling of TLR-7/8a to F trimers resulted in a concentration-dependent decrease in maximum binding to the following mAbs: AM14 (site V, prefusion-specific), D25 (site ⌀, prefusion-specific), MPE8 (site III, prefusion-specific), and 2374
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3, specific association between F-KSK and TRP-7/8a-ESE could be measured at both 30 and 37 °C (below and above the nanoparticle transition temperature). Greater maximum binding was observed at 37 °C, likely because of the greater size and mass of the bound nanoparticles compared to soluble polymer chains in a random coil conformation. Importantly, the interaction between the KSK and ESE coils was specific, as polymers bearing a control peptide sequence not predicted to form an α-helix did not significantly bind to F-KSK. Antigenic Characterization of RSV F Protein Coupled to TRP Nanoparticles. Although direct linkage of the TLR-7/ 8a to primary amines on F protein trimers was shown to reduce mAb binding to important neutralizing epitopes (Figure 1), we hypothesized that conjugation via coil motifs would facilitate its coupling to TRP-7/8a without interfering with neutralizing epitopes. We therefore assessed the binding of five mAbs (D25, 5C4, Motavizumab, MPE8, and AM14) with well-characterized and distinct neutralizing epitopes, to RSV F-KSK protein alone, or after coupling to TRP nanoparticles. Both the maximum binding and kon rates of F-KSK/TRP-7/8a-ESE were similar to F-KSK protein alone after nanoparticle formation at 37 °C, although there was less binding to some mAbs below the transition temperature (e.g., 5C4, 30 °C: ∼ 2-fold) (Figure 3c,d). These results show that modification of RSV F with a “KSK” coil to allow for site-selective coupling to TRP-7/8a-ESE nanoparticles preserves the structure of important epitopes on the immunogen. Immunogenicity of RSV F Protein Coupled to TRP Nanoparticles. We then compared immune responses following immunization of TRP nanoparticles co-delivering F protein and TLR-7/8a (F-KSK/TRP-7/8a-ESE) to soluble F with or without TLR-7/8a administered in a submicron polymer particle (PP-7/8a).20 Mice were immunized subcutaneously (SQ) at days 0 and 21 and serum assessed 10 days post-immunizations (Figure 4a). Both TLR-7/8a-containing formulations showed significantly enhanced prefusion F binding titers compared to that from soluble unadjuvanted F protein (Figure 4b). Similarly, these formulations induced a striking shift from TH2- to TH1- type antibody responses compared to F protein alone, as measured by F-specific antibody isotypes (Figure 4c). Analyses of draining lymph nodes after the boost revealed that, compared to results from F protein alone, immunization with both formulations of the TLR-7/8a increased frequencies of TFH cells, which are critical for antibody affinity maturation (Figure 4d). Because the RSV F protein immunogen is stabilized in the prefusion conformation, RSV F vaccine formulations should elicit prefusion-specific antibody responses and low-toundetectable postfusion-specific responses.3 Thus, the assessment of such responses provides a qualitative measure of the integrity of the stabilized RSV F trimers after formulation and immunization. Figure 4e shows that the addition of postfusion F (Post F) protein to sera competed only 26% of the binding activity against prefusion F (Pre F), demonstrating significant prefusion F-specific responses were induced. In contrast, serum binding to postfusion F protein was competed equally by prefusion and postfusion F protein, indicating responses to postfusion F were not specific to postfusion F but to epitopes shared by both forms. Furthermore, these serum responses were functional, as measured by an in vitro RSV neutralization assay (Figures 4f and S4). Of note, the F-KSK/TRP-7/8a-ESE formulation showed a consistent trend of higher reciprocal IC50-neutralizing activity compared to the admixed RSV F
Figure 2. Model for the coupling of protein immunogens with the thermoresponsive polymer delivery platform. Immunogens are recombinantly produced with a terminal α-helical coil peptide. Synthetic polymer chains are synthesized with hydrophilic and thermo-responsive blocks; TLR 7/8 agonists and complementary coil peptides are covalently attached to the hydrophilic block. Association of the protein immunogen with the polymer scaffold is driven by coil-coil heterodimer formation. After heating above a critical transition temperature, the thermoresponsive block collapses, excluding water. Multiple polymer chains are driven together by hydrophobic interactions, resulting in the formation of polymer particles with solution-exposed protein immunogens.
and “ESE” coil motifs, as no change in particle size was observed when a control peptide that is not predicted to form a coil motif was attached to TRP and formulated with RSV FKSK (Figure S3c). Pechar et al. previously used circular dichroism spectroscopy and analytical ultracentrifugation to characterize the interaction between analogous coil-forming peptides, which were used to link antibodies to a hydrophilic polymer,47 and we previously showed this strategy ensured co-delivery of HIV Gag-KSK with TRP-7/8a-ESE.20 However, to directly assess the ability of coil−coil interactions to couple RSV F protein to polymer nanoparticles, we measured the interaction between F-KSK and TRP-7/8a-ESE using BLI. As shown in panels a and b of Figure 2375
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Figure 3. RSV F trimers coupled to TRP particles via coil−coil interactions and retention of their antigenicity. (a) F-KSK was bound to Motavizumab-loaded BLI biosensors, then associated with TRP containing the complementary ESE coil (TRP-7/8a-ESE, blue traces) or a nonbinding control peptide (TRP-7/8a-control, red traces). Kinetic data were collected below the transition temperature (30°C), and after heating above the transition temperature (37°C). Colored traces raw data from serial dilutions (12.1-0.48 μM of peptide); black traces depict fitting data. (b) Binding affinity constants for the peptide interactions were derived from global fitting of the kinetic data at each temperature using a 1:1 Langmuir binding equation. (c,d) The RSV F mAbs 5C4, MPE8, Motavizumab, D25, and AM14 were loaded onto BLI biosensors and associated with F-KSK alone or in the presence of TRP-7/8a containing the complementary ESE coil (TRP-7/8a-ESE) or a nonbinding control peptide (TRP-7/8a control) in serial dilutions (162−40 nM of F protein). Kinetic data were collected at 30°C and 37°C. (c) Raw data (green traces) and fitting data (black traces) for binding to D25 are shown; vertical dotted lines indicate the transition from the association to dissociation phase. (d) Binding affinity constants were derived from global fitting of the kinetic data to each mAb at each temperature using a 1:1 Langmuir binding equation.
protein plus PP-7/8a formulation, suggesting that co-delivery of adjuvant and immunogen enhances the elicitation of neutralizing antibodies. Direct Coupling of RSV F Protein and TLR-7/8a. The above data show similar antibody titers following immunization with F-KSK/TPR-7/8a-ESE (that forms ∼40 nm sized particles) and F-KSK admixed with PP-7/8a (that forms ∼500 nm sized particles). Because these formulations have different particle sizes, which confound a direct comparison between co-delivery and admixing approaches, we directly addressed whether co-delivery of F protein and TLR-7/8a enhances antibody responses using only TRP particles. Accordingly, we compared three different formulations to FKSK alone: (1) F was delivered on a TRP particle without TLR-7/8a (F-KSK/TRP-ESE); (2) F was admixed with a TRP7/8a bearing a peptide that does not form a coil and therefore does not co-deliver both components (F-KSK/TRP-7/8acontrol); or (3) F was coupled to TRP-7/8a through coil− coil interactions (F-KSK/TRP-7/8a-ESE), packaging all components on a single particle. Mice were immunized and immune responses measured as described above (Figure 5a). After both the prime and the boost, F protein arrayed on
unadjuvanted TRP particles (F-KSK/TRP-ESE) elicited consistently higher titers than F protein alone, showing that multivalent immunogen arrays are more immunogenic than soluble proteins (Figure 5b). Among the groups receiving TLR7/8a, we noted that coupling of F to the TRP nanoparticles (FKSK/TRP-7/8a-ESE) elicited consistently higher binding titers after the prime and boost than F not coupled to TRP nanoparticles (F-KSK/TRP-7/8a-control), which were statistically higher than F-KSK alone (p > 0.001). Though co-delivery of TLR-7/8a and F protein was not required for the induction of TFH cells in the LN (Figure 5c), co-delivery induced consistently higher neutralization titers that were statistically higher than F protein alone (p > 0.05) (Figure 5d). Taken together, these data indicate that multimerizing the vaccine immunogen on particles and co-delivering potent immunostimulants, i.e., TLR-7/8a, are important components for maximizing the magnitude and neutralizing capacity of antibody responses. We next assessed how different routes of administration influenced antibody responses elicited by the formulation that induced the highest responses, F-KSK/TRP-7/8a-ESE. Intramuscular (IM) prime/boost was comparable to the SQ prime− 2376
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Figure 4. Particulate polymer adjuvants and improvement of antibody and T cell responses to RSV in mice. Immunization with F protein alone was compared to the addition of a TLR-7/8a containing polymer particle (PP-7/8a) or after coupling to TLR-7/8a containing TRP (TRP-7/8a-ESE). (a) Mice were immunized subcutaneously at days 0 and 21; serum and draining LN were collected at peak time points. (b) Anti-prefusion F binding titers were measured after the prime and boost by ELISA; dotted line indicates lower limit of detection. (c) TH1 or TH2 T skewing was assessed by measuring antibody isotypes IgG1 and IgG2a/c after the boost. The ratio of IgG1 to IgG2a/c is shown; bars indicate group geometric mean ratios. (d) TFH cells were measured in the LN as a percent of the CD44 hi cell population. (e) The specificity of serum from F-KSK / TRP-7/8a-ESE immunized mice was assayed by BLI. Serum binding to pre F (red) or post F (teal) was measured by competition with either pre F (dashed traces) or post F (dotted traces) compared to uncompeted sera (solid traces). Kinetic binding data from a single animal are shown (left plot); percent competition is graphed for all animals in the group (right plot). (f) In vitro RSV neutralization was measured by fluorescence reporter assay to obtain IC50 values. For comparison, the dotted line marks the clinical threshold for protection. For all panels, horizontal bars indicate group geometric means in log plots or group medians in linear plots. *, p < 0.05; **, p < 0.001; ***, p < 0.0001, compared to the “unadjuvanted F” group except where indicated otherwise. Data are representative of at least 2 independent mouse studies.
challenged intranasally (IN) with RSV A2 17 days after vaccination (Figure 6a). Consistent with the data shown above, antibody titers were highest when RSV F protein was coupled to TRP-7/8a (Figure 6b). Following challenge, unvaccinated mice showed RSV titers of ∼107 in the lung. However, the coupled F formulation (F-KSK/TRP-7/8a-ESE) resulted in complete protection, with viral titers below the limit of detection (Figure 6c). In contrast, two of five mice that received the uncoupled formulation (F-KSK/TRP-7/8a-control peptide) had RSV titers of 103 and 104, while the unadjuvanted TRP particles (F-KSK/TRP) had titers of ∼104. Anti(prefusion) F binding antibody titers strongly correlated with protection from challenge (Figure 6d), and flow cytometric analysis of lung-draining lymph nodes revealed that vaccination resulted in fewer DCs, with less co-stimulatory molecule expression (Figure 6e,f). These data suggest that the antibodies
boost immunization used throughout this study (Figure S5). Finally we investigated whether the activity of the TRP was dependent on the preformation of particles before administration or whether the TRP formulations could be kept in their soluble form until injection, whereupon particles would be formed after warming to 37 °C. A direct comparison between TRP formulations that were kept soluble prior to injection, and TRP particles preformed by heating before injection elicited comparable binding and neutralization titers (Figure S6). Because we observed that particle formation improves antibody titers (Figure 5), these data suggest that previously soluble TRP formulations undergo sufficient particle formation after injection in vivo. Coupled RSV F/TRP Formulation and Protection from RSV Challenge. To confirm that vaccine-elicited neutralizing responses could protect against a live viral challenge, mice were 2377
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Figure 5. Linking RSV F to polymer particles and improvement of immunogenicity. Immunization with soluble F-KSK was compared to F attached to unadjuvanted TRP (TRP-ESE), after attachment to adjuvant containing TRP (TRP-7/8a-ESE), or after mixing with adjuvanted TRP bearing a peptide that should not bind F-KSK (TRP-7/8a-control); formulations were pre-heated to 37 °C prior to injection. (a) Mice were immunized subcutaneously at days 0 and 21; serum and draining lymph nodes were collected at peak time points. (b) Anti-prefusion F binding titers were measured after the prime or boost by ELISA; dotted line indicates lower limit of detection. (c) TFH cells were measured in the LN as a percent of the CD44 hi cell population. (d) In vitro RSV neutralization was measured by fluorescence reporter assay to obtain IC50 values. The dotted line marks the clinical threshold for protection. For all panels, horizontal bars indicate group geometric means in log plots or group medians in linear plots. *, p < 0.05; **, p < 0.001, compared to the “F-KSK” group. Data are representative of at least 2 independent mouse studies.
adjuvants to maximize antibody titers. Another difference between TRP nanoparticles and soluble recombinant F protein53−55 or emulsion56 formulations is that TRP co-deliver the adjuvant and immunogen within the same particle. Using the facile process of coil−coil interactions, we show co-delivery is necessary for complete protection from RSV challenge in a mouse model (Figure 6c). Although other nanoparticle systems have been developed for co-delivery, including interbilayercross-linked multilamellar vesicles24 and various poly(lactide) (PLA) or poly(lactide-co-glycolide) (PLGA)-based nanoparticles,57,58 this is the first study to use site-specific linkers to codeliver a structurally optimized immunogen such as RSV F. Moreover, nanoparticles, VLPs and virosomes require complex cGMP purification processes and must be stable over time, while the encapsulation of antigens inside hydrophobic PLGA particles has been observed to cause protein instability.59 In contrast, TRP have the advantage of being soluble, filterable, colloidally dispersed, unimolecular polymers during purification and storage phases, which only transition into immunogenic nanoparticles after heating. Of note is the fact that the TRP formulation was equally immunogenic when particle formation was induced prior to or after injection, suggesting that preheating prior to injection is not required for optimal activity (Figure S6). We have shown how RSV F trimers can be attached to TRP by appending the KSK coil peptide at the protomers’ Cterminus. This coil−coil method60,61 may be broadly applicable for other immunogens designed to elicit neutralizing antibodies; however, other site-specific covalent approaches may also be suited, including chemical 62,63 and enzymatic approaches,64,65 as well as other high-affinity noncovalent interactions.66,67 However, the modularity and ease of the coil−coil approach make it translatable across many applications, such as for the attachment of multiple different immunogens from a given pathogen. For example RSV F, G, and M proteins could be co-delivered, which may provide improved protection.68,69 TRP nanoparticles can also be used
elicited by F-KSK/TRP-7/8a-ESE neutralized the viral inoculum, preventing infection and the onset of lung inflammation following challenge, highlighting the benefit of co-delivering immunogen and adjuvant on nanoparticles to maximize protective antibody responses.
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DISCUSSION The RSV F trimer immunogen has been structurally stabilized to elicit protective neutralizing responses yet still requires formulation and delivery with vaccine adjuvants to elicit high titers of neutralizing antibodies. However, a formulation of RSV F protein with adjuvants should not interfere with critical epitopes that can elicit neutralizing antibodies required for protection against RSV. Chemical conjugation is a common method to co-deliver proteins and TLRa and has been shown to elicit potent T cell responses.25−27,30,32 However, this approach may not be feasible with RSV F protein or other immunogens structurally optimized to elicit antibodies because of the propensity to disrupt neutralizing epitopes (Figure 1). Epitope occlusion is likely due either to steric hindrance by the TLRa or to the induction of protein aggregates, which were observed in some cross-linking conditions. Similar results have been reported with HIV Env,31 highlighting the need for a broadly applicable solution to couple TLRa to B cell immunogens that does not interfere with their antigenic surfaces. Here, we show that TRP nanoparticles are an effective platform for site-selectively coupling TLRa and RSV F trimers to achieve high titers of protective antibodies. There are several potential advantages that these nanoparticles have over soluble protein formulations. The first is the ability to multimerize protein subunits, which we demonstrate improves titers even without TLRa (Figure 5). Multimerization may also be achieved using self-assembling nanoparticles,7,8 virus-like particles (VLPs) and virosomes based on Newcastle disease virus,48 influenza,49,50 Lactococcus lactis,51 and RSV itself;52 however, these formulations may still require exogenous 2378
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Bioconjugate Chemistry
Figure 6. Co-delivery of RSV F protein and adjuvant and provision of protection from RSV challenge. (a) Mice were immunized subcutaneously at days 0 and 21; serum was collected at peak time points. Mice were challenged with RSV approximately 2.5 weeks after boosting; 4 days after challenge mice were sacrificed for analysis. Immunization with soluble F-KSK protein was compared to F-KSK attached to unadjuvanted TRP (TRPESE), after attachment to adjuvant containing TRP (TRP-7/8a-ESE), or after mixing with adjuvanted TRP bearing a peptide that should not bind FKSK (TRP-7/8a-control); formulations were pre-heated to 37 °C prior to injection. (b) Anti-prefusion F binding titers after boost; dotted line indicates lower limit of detection. (c) Viral lung titers were determined and graphed as pfu per gram of lung tissue; dotted line indicates lower limit of detection. (d) Correlation between peak F binding endpoint titers and viral lung titers; p-value derived with the Pearson test for correlation. After RSV challenge, mediastinal lymph nodes were measured for DC infiltration (e) and activation state (f). Migratory and CD8- population are shown as a percentage of non-B cells; activation is measured as MFI of CD80 expression. For all panels, horizontal bars indicate group geometric means in log ̈ group in (c,e). plots or group medians in linear plots. *, p < 0.05; **, p < 0.001; ***, p < 0.0001, compared to the F-KSK group in (b) or the naive Data are representative of at least 2 independent mouse studies.
to deliver synthetic peptide antigens.20 In this regard, vaccine design efforts have identified peptide B cell epitopes; for example, from HIV-1 Env,70,71 Burkholderia pseudomallei OppABP,72 and the malaria circumsporozoite protein,73 which could be multivalently arrayed on TRP to improve immunogenicity. Finally, multiple different adjuvants may also be co-delivered,20 potentially further boosting the magnitude or durability of antibody responses.57 For both adjuvant and synthetic peptide delivery, an array of chemical coupling methods, including azide chemistry, may be used for direct, covalent coupling to TRP chains.
The TLR-7/8a used here induces potent TH1 polarizing cytokines16,74 that promote TH1 RSV F-specific antibody responses (Figure 4c). While vaccine-induced TH1 immunity has been shown to be critical for protection against many pathogens,75 the most-common adjuvant, alum, induces TH2 polarized immunity.76 Studies have shown that TH2 immune responses induced by the failed FI-RSV vaccine38,39 were associated with vaccine-enhanced disease in children.40,77,78 Thus, TH1 type responses are especially desirable for an RSV ̈ infants. vaccine if the target population is antigen-naive Moreover, the use of TLR7a may be best suited to this 2379
DOI: 10.1021/acs.bioconjchem.6b00370 Bioconjugate Chem. 2016, 27, 2372−2385
Article
Bioconjugate Chemistry
H2O (2:1) solution and thoroughly bubbled with argon gas before the glass ampule reaction vessel was sealed and the reaction carried out at 70 °C for 18 h. The diblock polymer was isolated by precipitation to diethyl ether followed by reprecipitation from methanol to a 3:1 mixture of acetone and diethyl ether to yield 84.4 mg of the product. To remove the DTB end groups, the polymer and 12.9 mg of AIBN(0.79 μmol) were dissolved in 844 μL of DMF, and the solution was heated to 80 °C for 2 h. The resulting polymer was isolated by precipitation in diethyl ether and purified by gel filtration using a Sephadex LH-20 cartridge with methanol as the eluent yielding 72.4 mg of the product. The weight- and numberaverage molecular weights determined by SEC were Mw = 22 020 g/mol and Mn = 16 790 g/mol, respectively. The transition temperature (TT) of the polymer, determined by DLS, was 38 °C at 1.0 mg/mL 0.15 M PBS (pH 7.4). α-Helical Coil Sequences Used for Site-Specific Attachment. The following KSK coil sequence was appended to the RSV F protein: IAALKSK-IAALKSE-IAALKSKIAALKSK. The following sequences were attached to TRP: ESE coil, PEG4-IAALESE-IAALESE-IAALESK-IAALESECOOH; and control peptide, PEG4KPWPHEPRQVQGDNCRVNAKYLSVGEKY-COOH. Attachment of TLR-7/8a and ESE Coil to TRP. Different ligands (TLR-7/8a, ESE-coil peptide, control peptide, and fluorophore) functionalized with an azide group were attached to TRP through the propargyl side chain moieties distributed along the hydrophilic block A of the co-polymer by copper catalyzed 1,3 dipolar cycloaddition reaction. Reaction progress was monitored by HPLC. Example: A mixture of 20.0 mg of TRPP (7.1 μmol propargyl group), 1.0 mg of TLR-7/8a-azide (2.1 μmol), 0.4 mg of carboxyrhodamine 110-azide (0.7 μmol), 4.6 mg of ESE-coil peptide−azide (1.4 μmol) and 1.1 mg of TBTA (2.1 μmol) was dissolved in 460 μL of DMSO, and the solution was thoroughly bubbled with argon. Then, 0.84 mg of sodium ascorbate (4.2 μmol) in 168 μL of degassed water was added. Finally, a solution of 0.54 mg of CuSO4 in 108 μL of degassed water was pipetted to the reaction mixture to initiate the “click” reaction. The reaction was performed overnight at 45 °C until no unreacted ligands were detected by HPLC. The reaction mixture was diluted (1:1) with a saturated solution of EDTA in 0.15 M PBS (pH 7.4), and the conjugate was purified by gel filtration using a Sephadex PD-10 column with H2O as the eluent. The resulting conjugate was isolated from an aqueous solution by lyophilization, yielding 18.6 mg of the product. Determination of Polymer Molecular Weights. Molecular weights and polydispersities of polymers and co-polymers were measured by gel permeation chromatography using a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan) equipped with an internal UV−vis photodiode array (PDA) detector and external refractive index (RI) Optilab T-rEX and multiangle light scattering (MALS) DAWN HELEOS-II detectors (Wyatt Technology, Santa Barbara, CA). Either TSK-Gel SuperAW3000 and SuperAW4000 columns (Tosoh Bioscience, Japan) connected in series and 80% methanol−20% sodium acetate buffer (0.3 M, pH 6.5) as an eluent at a flow rate of 0.6 mL/min or a MicroSuperose 6 column (GE Healthcare) was used with PBS (pH 7.4) as the eluent at 0.1 mL min−1. A method based on the known total injected mass with an assumption of 100% recovery was used for the calculation of the molecular weights from light scattering data.
polarization, especially when vaccinating infants, who have restricted TLR responses.79 Overall, this study demonstrates that synthetic polymerbased nanoparticles are a flexible platform that achieves the codelivery of TLRa while preserving the antigenic surface of structurally stabilized B cell immunogens. Future studies with TRP nanoparticles will be undertaken to support HIV-1 Env SOSIP5,80 and peptide immunogens that have been structurally optimized to elicit protective antibody responses.
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EXPERIMENTAL PROCEDURES Chemicals. All chemicals were purchased from SigmaAldrich (St. Louis, MO) as reagent grade or higher purity, unless stated otherwise. Azido-PEG4-NHS and dibenzocyclooctyne (DBCO)-modified PEG spacer (DBCO-PEG4NHS) were purchased from Click Chemistry Tools (Scottsdale, AZ). Peptides were produced by solid-phase peptide synthesis and were obtained from American Peptide Company (Vista, CA). Synthesis of the imidazoquinoline-based TLR-7/8a, 1-(4(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4amine (2BXy), was synthesized as previously described.20,43,44 Direct Conjugation of TLR-7/8a to RSV F Using Primary Amine Chemistry. The TLR-7/8a, 2BXy, was coupled to the DS-Cav1 protein in a two-step reaction. First, the DS-Cav1 protein was functionalized with Azide groups through primary amines using either a 20:1 (hi) or 10:1 (lo) molar ratio of NHS-PEG4-Azide to DS-Cav1 in 100 mM HEPES buffer at pH 8.5. After 2 h, 20 or 10 equiv of DBCOmodified 2BXy (relative to molar equivalents of protein) were added, and then the reaction was permitted to proceed overnight at room temperature. To purify the protein from unreacted TLR-7/8a and linker, the reaction mixture was added to a Spectra/Por 7 dialysis tube (Spectrum Laboratories, Rancho Dominguez, CA) with a molecular weight cutoff of 25 kDa and dialyzed against PBS over 3 days. The resulting DSCav1-TLR-7/8a was analyzed by gel permeation chromatography using a TSK gel super SW3000 4.6 mm × 20 cm in a 2 μm column at 0.25 mL/min over 30 min in PBS, and TLR-7/ 8a content was determined by UV−vis spectrophotometry as described below. Diblock Co-polymer (TRP) Synthesis. Thermoresponsive polymer (TRP) nanoparticles were synthesized as previously described.20 Briefly, thermoresponsive A-B type diblock copolymers were prepared by RAFT polymerization in two synthetic steps. First, the hydrophilic block A was prepared by co-polymerizing N-(2-hydroxypropyl) methacrylamide (HPMA) with N-propargyl methacrylamide (PgMA) using 4,4′-azobis(4-cyanovaleric acid) (ACVA) as an initiator and 4cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTP) as a chain-transfer agent in molar ratios [M]/[CTP]/[ACVA] = 142:2:1 in 1,4-dioxane−H2O mixture. The reaction mixture was thoroughly bubbled with argon and polymerized in sealed glass ampules at 70 °C for 6 h. The resulting co-polymer was isolated by precipitation into a 3:1 mixture of acetone and diethyl ether and purified by gel filtration using a Sephadex LH-20 cartridge with methanol as the eluent. In the second step, the hydrophilic polymer block A bearing DTB terminal groups was subjected to a chain-extension polymerization through the RAFT mechanism with di(ethylene glycol) methyl ether methacrylate (DEGMA) to introduce the thermoresponsive polymer block B. A mixture of 50.0 mg of p[(HPMA)-co-(PgMA)] (5.31 μmol ∼ DTB gr), 53.0 mg of DEGMA (0.28 mmol), and 0.30 mg of ACVA (1.06 μmol) was dissolved in 477 μL of 1,4-dioxane/ 2380
DOI: 10.1021/acs.bioconjchem.6b00370 Bioconjugate Chem. 2016, 27, 2372−2385
Article
Bioconjugate Chemistry Determination of TLR-7/8a and Fluorophore Content on Polymers. The amount of TLR-7/8a or fluorophore attached to the co-polymers was determined by UV−vis spectroscopy, as previously described. Briefly, samples were suspended in solutions of 1% triethylamine−methanol at known concentrations and added to quartz cuvettes with a path length of 1 cm. Absorption was recorded over a spectrum from 250−775 nm using a Lambda25 UV−vis spectrophotometer from PerkinElmer (Waltham, MA). The amount of TLR-7/8a or fluorophore in solution was calculated from the Beer−Lambert law relationship. Hydrodynamic Size and Transition Temperature Determination. The hydrodynamic diameters (DH) and light scattering intensities (IS) of the polymers and co-polymers were measured by the DLS technique at a scattering angle θ = 173° using a Nano-ZS instrument (model ZEN3600, Malvern Instruments, Malvern, England) equipped with a 632.8 nm laser. The temperature measurements were performed to investigate the self-assembly of polymer coils to polymer micelles in the temperature range 20−50 °C (in 1 °C increments) in PBS (1.0 mg/mL, pH 7.4) solutions. At each step, measurements were performed after reaching steady-state conditions, which typically required approximately 10 min. For the evaluation of the dynamic light scattering data, the DTS(Nano) program was used. The mean of at least three independent measurements was calculated. The transition temperature (Ttr) characterizing the polymer chain conformational changes was evaluated from the temperature dependence of the hydrodynamic diameter (DH); the Ttr value was determined from the intersection point of two lines formed by the linear regression of a lower horizontal asymptote and a vertical section of the S-shaped curve (sigmoidal curve) fit. RSV F Protein and Immunogen Constructs. DS-Cav1 and postfusion constructs are as previously described.35,81 The KSK coil sequence and glycine−serine linker (GGGSGGGSGG) were introduced at the C-terminus of the DS-Cav1 construct by site-directed mutagenesis. RSV F variants were produced as previously described.35 In brief, variants were expressed by transient transfection of Expi293F cells using 293Fectin (Invitrogen). Cell culture supernatants were harvested 5 days post-transfection and centrifuged at 10000g to remove cell debris. The supernatants were sterilefiltered, and RSV F proteins were purified by nickel and streptactin-affinity chromatography followed by size-exclusion chromatography. Vaccine Formulation. Polymer-based adjuvants were diluted in PBS and normalized so the TLR-7/8a agonist dose was equal across all vaccine groups. For thermoresponsive polymer (TRP) constructs, polymers bearing the ESE or control peptides were mixed with RSV F-KSK coil protein for at least 30 min prior to injection. 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DOI: 10.1021/acs.bioconjchem.6b00370 Bioconjugate Chem. 2016, 27, 2372−2385
