PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine

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PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine – Self-Assembled Supramolecular Complexes Enable Enhanced Immunogenicity and Protection Corinne Cayatte, Alexander Marin, Gaurav Manohar Rajani, Kirsten SchneiderOhrum, Angie Snell Bennett, Jason D. Marshall, and Alexander K. Andrianov Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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Molecular Pharmaceutics

PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine – Self-Assembled Supramolecular Complexes Enable Enhanced Immunogenicity and Protection Corinne Cayatte,† Alexander Marin,‡ Gaurav Manohar Rajani,† Kirsten Schneider-Ohrum,† Angie Snell Bennett,† Jason D. Marshall,†,§ and Alexander K. Andrianov*,‡ † Infectious Disease/Vaccines Department, MedImmune, Gaithersburg, Maryland 20878, United States ‡ Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850, United States KEYWORDS: polyphosphazenes, immunoadjuvants, self-assembly, vaccines, respiratory syncytial virus, vaccine delivery, complexation

ABSTRACT: PCPP, a well-defined polyphosphazene macromolecule, has been studied as an immunoadjuvant for a soluble form of the post-fusion glycoprotein of respiratory syncytial virus (RSV sF), which is an attractive vaccine candidate for inducing RSV-specific immunity in mice and humans. We demonstrate that RSV sF-PCPP formulations induce high neutralization titers to

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RSV comparable to Alum formulations even at a low PCPP dose and protect animals against viral challenge both in the lung and upper respiratory tract. PCPP formulations were also characterized by Th1-biased responses, compared to Th2-biased responses that are more typical for RSV sF alone or RSV sF-Alum formulations, suggesting an inherent immunostimulating activity of the polyphosphazene adjuvant. We defined these immunologically active RSV sFPCPP formulations as self-assembled water-soluble protein-polymer complexes with distinct physico-chemical parameters. The secondary structure and antigenicity of the protein in the complex were fully preserved during the spontaneous aqueous self-assembly process. These findings further advance the concept of polyphosphazene immunoadjuvants as unique dualfunctionality adjuvants integrating delivery and immunostimulating modalities in one watersoluble molecule.

1. INTRODUCTION Poly[di(carboxylatophenoxy)phosphazene], PCPP is a well-defined macromolecule (Chart 1), which has displayed potent immunoadjuvant effect when administered with a variety of bacterial and viral antigens in vivo.1-3 A biodegradable synthetic molecule, it is characterized by an excellent water-solubility that allows for straightforward formulation approaches and improved vaccine stability.4, 5 As an immunoadjuvant, PCPP displays dual functionality serving both as vaccine delivery vehicle and immunostimulant.6-10 This has been linked to the ability of polyphosphazene to spontaneously self-assemble with vaccine antigens and toll-like receptors.6, 10

Clinical trials have documented that PCPP-adjuvanted vaccines exhibit favorable safety and

immunogenicity profiles.11-13 Other representatives of the polyphosphazene immunoadjuvant


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family have also been synthesized and their physico-chemical characteristics and biological potencies were found to be strongly dependent on molecular structure and composition of the polymer.14, 15 Infections associated with Respiratory Syncytial Virus (RSV) remain an unmet medical challenge in both full-term infants and older adults, but the development of a safe and effective vaccine has proven difficult.11 To this end, PCPP demonstrated substantial promise as an adjuvant for a co-purified RSV F, G, and M subunit vaccine in two phase I clinical trials, in which neutralizing antibodies were used as clinical endpoints. In these studies, PCPP formulations elicited an equal to or greater than 4-fold rise in neutralizing antibodies against RSV A or RSV B in > 75% of the participants.11 It was also noted that the same vaccine adjuvanted with Alum yielded comparable results.11 A different polyphosphazene adjuvant, PCEP, has also shown significant potential with RSV antigen in vivo especially when formulated in combinations with other adjuvants or in a microparticulate form.16-20 However, except for the above-mentioned synopsis of clinical data, no data on in vivo or in vitro properties of RSV-PCPP vaccine formulations have been published. Recent studies on physico-chemical characterization of polyphosphazene-adjuvanted vaccine formulations emphasized the importance of non-covalent interactions between vaccine antigens and PCPP.6 The advancement of well-defined recombinant antigens and sophisticated analytical approaches provides new insights and can result in new methodologies for optimizing polyphosphazene formulations.6 Therefore, in order to develop a successful PCPP-adjuvanted vaccine, it is imperative to understand the potential role of such interactions, investigate and optimize physico-chemical parameters and antigenicity of resulting non-covalent complexes, and evaluate the in vivo performance of such formulations in the context of the above properties.


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In the present paper, we conducted a comprehensive physico-chemical and in vivo investigation of RSV sF-PCPP vaccine and benchmarked its key immunological parameters against RSV-Alum formulations in mice. In this study, we employed a soluble form of the recombinant post-fusion glycoprotein (RSV sF) of RSV, which plays a critical role in RSV infection and is an advantageous vaccine candidate for inducing RSV-specific immunity21, 22 as the antigenic component of the vaccine. For the first time, we define immunologically active RSV sF-PCPP formulations as self-assembled water-soluble protein-polymer complexes with distinct physico-chemical parameters. We report that only PCPP-based, but not Alum-based, formulations were capable of fully protecting animals against RSV challenge, both in the lung and the upper respiratory tract. 2. MATERIALS AND METHODS 2.1. Materials. Poly[di(carboxylatophenoxy)phosphazene], PCPP was synthesized, purified, and characterized as described previously.23, 24 Alum - Alhydrogel (Aluminum hydroxide) adjuvant (Brenntag, Frederikssund, Denmark), Phosphate Buffered Saline (PBS, pH 7.4) (Thermo-Fisher Scientific, Grand Island, NY, USA) were used as received. 2.2. Dynamic Light Scattering (DLS). Molecular size distribution profiles of PCPP and its formulations with RSV in aqueous solutions were obtained using ZetaSizer Nano series, ZEN3500, (Malvern Instruments Ltd., Worcestershire, UK). Formulation components were filtered using 0.22 µm Millex syringe filters (EMD Millipore, Billerica, MA) before mixing. 2.3. Asymmetrical Flow Field Flow Fractionation (AF4). Composition analysis of RSVPCPP interactions was conducted by Asymmetrical Flow Field Flow Fractionation (AF4), which offers the possibility of a separation under shear free conditions and without unwanted


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interactions with the surface of a stationary phase.25 AF4 is an elution-based method, in which the separation is carried out in a single liquid phase and an external flow of the mobile phase is applied perpendicularly to the direction of sample flow through a channel equipped with semipermeable membrane. Similar to size-exclusion HPLC, the materials are separated by size, however, as opposed to chromatographic methods, the upper size limit for the analyte can reach as high as 100 µm. Formulations were dissolved in 1xPBS (pH 7.4), which was also used as an eluent. The size-dependent diffusion abilities of macromolecules lead to an arrangement in different layers of the parabolic flow profile inside the channel with small molecules eluting first and the larger molecules or particles eluting later.25 The AF4 system, a Postnova AF2000 MT series (Postnova Analytics GmbH, Landsberg, Germany), was equipped with two PN1130 isocratic pumps, PN7520 solvent degasser, PN5120 injection bracket and UV-Vis detector (SPD20A/20AV, Shimadzu Scientific Instruments, Columbia, MD 21046). A regenerated cellulose membrane with molecular weight cutoff of 10 kDa (Postnova Analytics GmbH, Landsberg, Germany) and a 350 µm spacer were used in a separation micro-channel employing both laminar and cross flows of an eluent - PBS (pH 7.4). The collected data was processed using AF2000 software (Postnova Analytics GmbH). 2.4. Circular Dichroism Spectroscopy. Circular dichroism (CD) measurements were carried out using a Chiroscan qCD spectrometer (Applied Photophysics, Leatherhead Surrey, United Kingdom) in a 1 mm cell at ambient temperature. 2.5. PCPP-RSV sF ELISA binding. High-binding 96-well plates were coated with RSV sF at 1.5 µg/well with or without pre-mixing with increasing concentrations of PCPP (0.75, 2.5, 10, 25, and 50 µg). RSV sF specific antibodies (Motavizumab, 1331H and 43) were serially diluted by 3-fold increments starting from 1 µg/mL in sample diluent (PBS with 1% BSA and 0.05%


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Tween 20). Bound antibodies were detected using HRP-coupled rabbit anti-human IgG antibodies for Motavizumab and 43, and HRP-coupled goat anti-mouse IgG for 1331H (both secondary antibodies were from Dako). 2.6. Animal studies. Pathogen-free, 6-7 week-old BALB/c mice were purchased from Harlan (Indianapolis, IN). All animal procedures were conducted according to the guidelines of the MedImmune Institutional Animal Care and Use Committee (IACUC). The soluble post-fusion (RSV sF) antigen was expressed in the Chinese Hamster Ovary (CHO) cell line (ATCC, Manassas, VA) using a fed-batch manufacturing process and was purified using a 3-step chromatography process, a nanofiltration virus removal step, and a low pH virus inactivation step. PCPP formulations were prepared on the day of injection. A solution of PCPP in PBS was filtered through 0.22 µm Millex GV sterile filter and then mixed with solution of RSV-sF at 1:1 ratio by volume. Formulations were vortexed for 15- 20 seconds and then equilibrated at ambient temperature for approximately one hour. Alum was adsorbed to protein by mixing O.N. at 4°C. The dose of adjuvant (100 µg) was chosen based on previous investigations with RSV and general guidelines for preclinical studies.22, 26-29 Mice were randomly assigned into groups (n=5 mice per group), primed at day 0, and boosted at day 21 intramuscularly (i.m.) with 1.5 µg of RSV sF and different adjuvant preparations. Placebo groups were immunized with PBS. The RSV infection control group was infected intranasally (i.n.) at day 0 with 106 PFU of wild-type (wt) RSV A2. All mice were challenged i.n. at day 35 with 106 PFU of RSV A2. Mice were retro-orbitally bled at day 35 prior to viral challenge for neutralizing antibody titer evaluation. All animals were euthanized at day 39, blood was harvested for serum IgG isotyping, and spleens were harvested for analysis of cellular immune responses, and lungs and nasal turbinates


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for RSV A2 titer measurement by plaque assay and IFN-γ and IL-5 expression in lung supernatant. 2.7. Micro-neutralization assay. Serum samples collected prior to challenge (day 35) were heat-inactivated at 56°C for 45 min. In 96-well plates, the positive control antibody (palivizumab) was serially diluted by 3-fold increments (starting at 8 µg/mL) in cell culture media (minimal essential medium (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U of penicillin/mL, and 100 µg of streptomycin/mL (all from Invitrogen)) for a final volume of 50 µL. In duplicate, the test sera (starting dilution 1:2) were serially diluted by 3-fold increments in cell culture media for a final volume of 50 µL. Each serum dilution was mixed with 50 µL RSV A2 at 500 PFU per well. Following 2 h incubation at 37°C with 5% CO2, 2.5 × 104 HEp-2 cells in 100 µL volume were added to each well. Cells plus virus and cells-only wells served as controls. After 3 days of incubation at 37°C with 5% CO2, the cell culture medium was removed and the monolayer was fixed with chilled 80% acetone. RSV replication was visualized by immunostaining with an HRP-labeled 1331H monoclonal anti-RSV F antibody. The reciprocal log2 of the IC50 was determined for each serum sample using Prism GraphPad software. The lower limit of detection (LLOD) of this assay is 4 log2. If an IC50 value could not be calculated, the log2 of the initial dilution (1:16) was used for analysis. 2.8. Serum IgG, IgG1, IgG2a ELISA. RSV sF-specific IgG antibody titers were measured in serum at the day of sacrifice (day 39). High-binding 96-well plates were coated with RSV sF at 100 ng/well. Control antibodies (purified 1331H for total IgG and IgG2a and purified 1308F for IgG1)30 were serially diluted by 3-fold increments starting from a concentration of 1 µg/mL in sample diluent (PBS with 1% BSA and 0.05% Tween 20). Samples were diluted in diluent at 1:100 for non-immunized animals, at 1:105 and 1:106 for RSV A2-immunized animals, and at


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1:106 and 1:107 for RSV sF + adjuvant-immunized animals. Bound total IgG, IgG1 or IgG2a were detected with the appropriate HRP-labeled antibody (goat anti-mouse IgG from Dako, and goat anti-mouse IgG1 and IgG2a antibodies from Jackson ImmunoResearch Laboratories, Inc). The serum antibody titers were calculated based on the standard curves to determine µg/ml of each antibody type (SoftMax Pro 5.4). 2.9. Cell mediated immunity. Splenocytes were isolated as previously described.31 The number of mouse splenocytes secreting gamma interferon (IFN-γ) was determined by enzymelinked immunospot (ELISPOT) assay (BD Biosciences, San Diego, CA) according to the manufacturer's recommendations. For the in vitro stimulation, splenocytes from individual mice (5×105/well) were incubated with RSV F specific CD4 peptide (GWYTSVITIELSNIKE),32 or with a H-2kd-specific CD8 peptide (KYKNAVTEL) 33 (JPT, Berlin, Germany) at a concentration of 1 µg/mL. Controls included splenocytes that were stimulated with Cell Stimulation Cocktail (eBioscience, San Diego, CA) or mock-stimulated. Following 20 h of incubation in the presence of peptide at 37°C in a humidified incubator, the ELISPOT assay was completed and spots were counted by an AID ELISPOT assay reader (Autoimmune Diagnostica, Germany). For analysis, the spot counts in medium control wells were subtracted from the specific spot count after peptide stimulation, and the difference is reported as the number of spotforming cells (SFC) per 1 × 106 splenocytes. 2.10. Cytokine quantification. Lung-clarified homogenates collected 4 days post-RSV A2 challenge were each evaluated for the presence of IFN-γ and IL-5 cytokines in a Luminex-based cytokine profiling assay (Millipore, Billerica, MA) according to the manufacturer protocol and analyzed using a Bio-Rad Luminex 200 reader (Bio-Rad, Hercules, CA).


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2.11. Viral plaque assay. Lungs and nasal turbinates were placed in cold Hanks balanced salt solution (Thermo Fisher, Waltham, MA) supplemented with 1X sucrose phosphate (HyClone, GE Healthcare Lifesciences, Pittsburgh, PA) in a tissue homogenization tube (MP Biomedicals, Santa Ana, CA) and processed using an MP FastPrep24 instrument (MP Biomedicals). Clarified supernatants were serially diluted and applied onto sub-confluent HEp-2 cells in 24-well plates. After 90 min of incubation, supernatants were removed and cells were overlaid with MEM+FBS+Pen/Strep supplemented with 0.75% methylcellulose. After 5 days, cells were fixed with 100% methanol for 15 min. Following fixation, cells were blocked for 1 h in 5% nonfat milk and stained for 1 h with a RSV goat polyclonal antibody (Chemicon) followed by staining for 1 h with a HRP-conjugated rabbit anti-goat antibody (DAKO). To visualize plaques, cells were incubated with AEC substrate ready-to-use (DAKO) followed by a water rinsing step. PFU/mL were calculated based on number of plaques and dilution factors. 3. RESULTS 3.1. Self-Assembly in PCPP – RSV Formulations. The ability of PCPP to spontaneously self-assemble with proteins in aqueous solutions6 requires a thorough investigation of their formulations to detect and characterize potential antigen-adjuvant complexation in the system. Although the existence of such interactions is frequently ignored, under certain circumstances they can lead to conformational changes in the polymer, which can potentially affect immunogenicity of formulations in vivo.10 Therefore, RSV sF-PCPP formulations were analyzed by the dynamic light scattering (DLS) method to detect any alterations in molecular sizes and conformations of polyelectrolyte – PCPP.34, 35


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Addition of RSV sF to PCPP in 1xPBS increased the hydrodynamic diameter of the polymer as determined by DLS, clearly demonstrating self-assembly of antigen and adjuvant in the system (Figure 1A). Interestingly, an increase in the ionic strength of the buffer can potentially reverse this effect with the dimensions of RSV sF-PCPP in 3xPBS being practically equal to those of PCPP without RSV sF in 1xPBS (Figure 1B). This indicates either a more compact conformation of the complex at a higher salt concentration, which has been previously reported for PCPP,23 or interference of sodium chloride with complex formation. The addition of another common formulation excipient, histidine, to the PCPP-RSV sF formulation in 1x PBS resulted in further increase of complex dimensions, suggesting that basic formulation components can further facilitate complex formation (Figure 1C). In the development of formulation protocols for this study, we also evaluated the effect of antigen-adjuvant concentrations and mixing ratios. These studies revealed that the use of small volumes of concentrated antigen stock solution upon mixing with PCPP resulted in the formation of large insoluble aggregates (Figure 1D). Since such heterogeneous formulations can lead to non-reproducible results, care was taken to employ more diluted antigen solutions and monitoring formulations by DLS. Overall, DLS data unambiguously demonstrated spontaneous formation of PCPP-RSV sF supramolecular complexes and the ability to modulate interactions through variation in the ionic environment. Importantly, no reduction in PCPP molecular size was observed, which has been previously shown to be detrimental to the immunogenicity of the complex.10 3.2. Composition Analysis of RSV sF-PCPP Complexes. Compositions of antigen-polymer supramolecular complexes were studied using an asymmetric flow field flow fractionation (AF4)


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method with 1xPBS as a mobile phase. AF4 is an elution-based method, in which the separation is carried out in a single liquid phase and an external flow of the mobile phase is applied perpendicularly to the direction of sample flow through a channel equipped with a semipermeable membrane.25 Similar to size-exclusion HPLC, the materials are separated by size, however, as opposed to chromatographic methods, the upper size limit for the analyte can reach as high as 100 µm.25 The method minimizes or even completely eliminates one of the main experimental challenges in a direct investigation of molecular interactions, such as potential interference with stationary phase (HPLC or Surface Plasmon resonance methods), or the influence of the electric field (electrophoretic methods).25 Representative examples of AF4 fractograms for RSV sF, PCPP, and RSV sF-PCPP formulation are shown in Figure 2A. As seen from the Figure, the protein peak (17 minutes), which is clearly visible in the RSV sF elution profile, is not observed in the AF4 trace of the complex. In fact, the UV absorption at this elution time is practically identical for PCPP and PCPP-RSV sF profiles. This provides evidence of antigen-PCPP complexation and the absence of ‘unbound’ RSV sF in the formulation. Moreover, there is a corresponding increase in the area of the ‘macromolecular’ peak (19-24 minutes) in the RSV sF-PCPP formulation and the peak shift towards larger sizes compared to the PCPP profile. This confirms the association of protein with polyphosphazene and correlates well with DLS data on the self-assembly in the RSV sFPCPP system. AF4 method was further employed for analyzing formulations with varying protein-polymer ratios. The binding isotherm for RSV sF-PCPP formulations is shown in Figure 2B. As seen from the Figure, the saturation of PCPP with RSV sF in the complex is achieved at very low concentrations of RSV sF. From a practical standpoint, it was also important to evaluate the


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percent of bound antigen as a function of PCPP-to-RSV sF ratio. Figure 2C shows that practically 100 % of RSV sF is bound to PCPP in all formulations in which the antigen to polymer ratio does not exceed 5:1 by weight.

3.3. Structural Integrity and Antigenicity of RSV-PCPP Formulations in Vitro. The existence of distinct interactions in RSV sF-polyphosphazene formulations necessitated further in vitro investigations to demonstrate functional viability of the antigen despite formation of noncovalent complexes with polyphosphazene. This was especially important as the formation of ‘compact’ complexes can lead to some loss of immunogenicity in vivo.10 Conformational stability of RSV sF in a non-covalent complex with PCPP was evaluated using circular dichroism (CD) spectroscopy. No loss of the native fold of the RSV sF protein was detected upon addition of polyphosphazene at various concentrations (Figure 3) indicating that the protein maintains its structural integrity in the complex with PCPP. The antigenicity of PCPP-adjuvanted RSV sF formulations was evaluated in vitro using ELISA assays with antibodies targeting 3 different epitopes – Motavizumab (site A), 1331H (site C), and 43 mAbs. 30, 36, 37 Antigen to PCPP ratio was varied by altering the polymer concentration in formulations. Figure 4 demonstrates that PCPP only had a marginal effect on the availability of the antigen as detected by the assay. The results were similar for all antibodies and the effect of polyphosphazene was practically negligible at high dilutions. This suggests, that despite the formation of the complex, the antigen remains largely accessible to antibodies, which allows further investigation of polyphosphazene formulations in vivo.


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3.4. In vivo Immunogenicity Studies. In order to evaluate the immunogenicity of PCPPadjuvanted formulations, we used RSV sF as a model antigen in the BALB/c mouse strain which is semi-permissive to RSV A2 challenge.38 RSV sF alone usually induces low neutralizing antibody titers, a Th2-biased immune response, very low cell-mediated immunity, and partial protection from challenge in the lungs at low antigen doses.22, 26, 39 Formulating with Alum greatly enhances the neutralization titers, improving the lung protection from viral challenge, but maintaining a Th2 bias.22, 26 Here, we wanted to compare PCPP-RSV sF formulations to RSV sF-Alum and natural infection. BALB/c mice were immunized i.m. at day 0 and day 21 with increasing doses of PCPP (5, 25, and 50 µg) formulated with a fixed dose of 1.5 µg of RSV sF, with RSV sF-Alum formulation (100 µg alum with 1.5 µg RSV sF), or i.n. immunized with wt RSV A2. Humoral response analysis (Figure 5) showed that all RSV sF-adjuvanted formulations induced statistically significantly higher titers than live RSV infection (Figure 5A). PCPP dosedependent effect was observed on neutralizing antibody titers, RSV F specific IgG total (Figure 5B), IgG1 and IgG2a (Figure 5C). Even at the lowest PCPP dose tested (5 µg), PCPP-RSV sF formulation induced high neutralization titers that were comparable to titers induced by RSV sF formulated with 100 µg of Alum. IFN-γ promotes class-switching from IgG1 to IgG2a in the mouse, making IgG2a titers a correlate for Th1 bias. Here, we observed a dose-dependent effect of PCPP on anti-RSV IgG2a isotype levels leading to a balanced IgG1/IgG2a response at 50 µg of PCPP-RSV sF as opposed to a strongly skewed IgG1 response observed in the Alum-RSV sF immunized group (Figure 5C).


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We then evaluated the ability of PCPP to prime for RSV F-specific CD4 and CD8 T cell responses (Figure 6). Splenocytes were stimulated in vitro with RSV F-specific peptides representing CD4 or CD8 T cell epitopes (Figure 6A and B) and the number of IFN-γ-producing cells per 1x106 splenocytes was evaluated by ELISPOT assay. Both CD4 and CD8 T cell responses plateaued at 25 µg of PCPP reaching levels comparable to those induced by Alum adjuvant. CD8 responses induced by 25 and 50 µg of PCPP were statistically higher than Fspecific responses induced by live virus immunization. Lastly, we evaluated the Th1/Th2 expressed cytokine ratio as well as RSV viral loads in the respiratory tract from animals challenged i.n. with wt RSV A2 (Figure 7). The lung cytokine profile showed a shift towards a Th1 bias with the downregulation of IL-5 and IL-13 when PCPP doses were increased to 25 and 50 µg (Figure 7A and B). All formulations tested conferred full protection from RSV infection in the lungs (Figure 7C) and increasing doses of PCPP improved the protection from challenge in nasal turbinates up to complete protection with 50 µg of RSV sF-PCPP (Figure 7D), contrasting with Alum which failed to induce full protection in the upper respiratory tract. 4. Discussion PCPP adjuvant has been the focus of extensive in vivo research studies with multiple viral and bacterial vaccine antigens.2, 40 Despite reports that indicated polyphosphazene polyelectrolytes can spontaneously self-assemble into supramolecular complexes with proteins and antigens,6, 7, 10 the vast majority of publications on polyphosphazene adjuvants appear to ignore this critical physico-chemical parameter and its potential influence on the immunogenicity of formulations and reproducibility of the results. Physico-chemical characterization of RSV sF-PCPP


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formulations by DLS and AF4 methods unambiguously demonstrate spontaneous self-assembly of antigen with the polymer resulting in the formation of water-soluble supramolecular complexes, which typically exceed the molecular size of the polymer alone. Quantitative analysis using AF4 method also reveals that RSV sF remains bound to PCPP even in formulations with a molar excess of the antigen. This allows for multimeric antigen complexes, in which PCPP acts as a scaffold for displaying multiple copies of RSV-F and suggests the presence of strong interactions in the system. Generally, for a reaction between macromolecule with identical binding sites and the ligand, the dissociation constant can be conveniently estimated as a concentration of ligand at a half-saturation point.41 If this simplified approach is applied to data presented in Figure 2B, the value of the apparent dissociation constant lies in the sub-micromolar range, which suggests strong avidity between RSV-F and PCPP. Although it is not possible to make an accurate comparison of the above apparent dissociation constant with values for other biological systems due to the simplified nature of the approximation, it still may be worth mentioning that the micromolar range of dissociation constants is typical for binding of signaling protein to a biological target.42 The existence of strong intermolecular interactions in polyphosphazene-adjuvanted vaccines emphasizes the need for adequate analytical support of formulation development. Despite evident water-solubility of the majority of RSV-PCPP complexes, certain formulation conditions can potentially lead to severe aggregation, potentially unbound PCPP, and generally result in undesirable heterogeneous systems (Figure 1D). This can be explained in view of rapid kinetics of complex formation and its high stability, which are characteristics of strong intermolecular binding. Similarly to other macromolecular exchange reactions involving polyelectrolyte systems,43 strong interactions may hinder antigen molecules from distributing homogeneously


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among polymer chains. Although typically not detrimental, these effects can become pronounced when small volumes of highly concentrated antigen stock solutions are used (Figure 1D). In such cases, the formation of supramolecular complexes saturated with the excess of antigen may result in compact polymer conformation and eventual loss of water-solubility of the complex.10 Once again, this emphasizes the importance of physico-chemical characterization of polyphosphazene formulations, which is often neglected. Despite the presence of strong interactions in PCPP-RSV sF system, CD spectroscopy did not detect any changes compared to antigen alone, which suggests that RSV sF antigen in the watersoluble complex remains structurally unchanged (Figure 3). Moreover, RSV sF antigen in its formulation with PCPP remains largely accessible to three different antibodies (Figure 4). The immunogenicity of PCPP formulations in mice was investigated in respect to the induction of antibody responses, cell mediated immunity, and protection against challenge. All polyphosphazene formulations displayed high potency in the induction of humoral responses, to levels that were higher than levels we typically observed following wt RSV A2 infection.39, 44 Five µg of PCPP, the minimal dose of polyphosphazene adjuvant tested in this study, was as efficient in enhancing neutralization titers to RSV sF as 100 µg of Alum. PCPP dose-dependency of the response is especially evident from the data on specific anti-RSV F total IgG, IgG1, and IgG2a titers. Somewhat unexpectedly, polyphosphazene formulations demonstrated the ability to induce a Th1-biased response in the absence of a strong immunopotentiator, such as toll-like receptor (TLR) agonist. This is indicated by the ratio of IFN-γ/IL-5 production and low levels for IL-13 present in lungs post-RSV A2 challenge. Furthermore, a balanced IgG1/IgG2a response, yet


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another correlate of Th1/Th2 response, was observed for a formulation with high dose of PCPP. The results for PCPP formulations contrast with the Th2-biased response observed with RSV sF alone or RSV sF-Alum formulations.22, 26, 39 These observations can be potentially explained by recent findings on the multifunctional nature of polyphosphazenes.6 In addition to antigen delivery modality, PCPP has been shown to be capable of interacting with TLRs in solution and activating cells with overexpressed TLRs.6 Finally, perhaps one of the most important outcomes of this study is that despite similar neutralization titers induced by PCPP and Alum adjuvanted formulations, only PCPP, but not Alum at the dose employed, has been able to demonstrate complete protection of animals against challenge at the doses of antigen and Alum employed in the study. Conclusions The present study demonstrates that PCPP, a water-soluble synthetic macromolecule of the polyphosphazene family, displays high immunoadjuvant potency when formulated with RSV sF antigen. PCPP formulations induce high neutralization titers similar to Alum formulations, and stimulate Th1 biased response. While Alum protected animals against challenge in the lung, PCPP formulation provided complete protection against challenge both in the lung and the upper respiratory tract. Evaluating PCPP immunogenicity in combination with the pre-fusion form of the F antigen might be of interest, given the potent elicitation of neutralization titers induced by this antigen described in the literature.45 We found clear evidence that PCPP can act as an antigen delivery vehicle. Physico-chemical methods prove spontaneous self-assembly of RSV sF with polyphosphazene into non-covalent macromolecular complexes. Present results indicate that, in a majority of formulations, such


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interactions do not adversely affect secondary structure and antigenicity of the protein. However, it was also found that the formation of undesirable heterogeneous and aggregated systems could potentially occur, especially when highly concentrated stock solutions of PCPP and antigen are employed. Therefore such conditions should be avoided and physico-chemical methods, such as DLS and AF4, should be implemented in the formulation efforts to provide for predictable results and their reproducibility. Finally, the Th1 biased response observed in this study for PCPP formulations, as opposed to Th2-biased response, which is typical for RSV sF alone or RSV sF-Alum formulations, emphasizes the importance of our still evolving understanding of PCPP role as immunostimulant. This further advances the concept of polyphosphazene immunoadjuvants as unique dual-functionality adjuvants integrating delivery and immunostimulating modalities in one water-soluble molecule. Chart 1. Chemical structure of PCPP, sodium salt. Figure Captions Figure 1. DLS profiles for RSV sF-PCPP formulations in (A) PBS, (B) PBS of various ionic strength, (C) PBS and histidine buffer (0.1 mg/mL PCPP, 0.05 mg/mL RSV sF), and (D) RSV sF-PCPP formulations prepared using 1:6 and 2:1 mixing ratios of RSV sF and PCPP stock solutions, volumetric ratio - VRSV:VPCPP, PBS (0.05 mg/mL PCPP, 0.1 mg/mL RSV sF). Figure 2. (A) AF4 profiles for RSV sF-PCPP formulations (0.5 mg/mL PCPP, 0.2 mg/mL RSV sF, 280 nm, PBS), (B) binding isotherm for RSV sF-PCPP formulations (3 µg/mL PCPP, 0-300


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µg/mL RSV sF, PBS) and (C) the percent of bound RSV vs. PCPP:RSV sF ratio by weight (PBS). Figure 3. Circular dichroism spectra of RSV sF in the presence of various concentration of PCPP (0.1 mg/mL RSV sF, PBS). Figure 4. Antigenicity of PCPP-adjuvanted formulations in vitro using ELISA assay with Motavizumab, 1331H, and 43 mAbs (PCPP concentrations – 0 (1), 0.0075 (2), 0.025 (3), 0.1 (4), 0.25 (5) and 0.5 mg/mL (6); RSV sF concentration – 0.015 mg/mL). Figure 5. Antibody responses induced by increasing doses of PCPP. Animals (n=5 per group) were immunized i.m. with RSV sF formulated with 5, 25, or 50 µg of PCPP or 100 µg of Alum at day 0 and day 21 and challenged i.n. at day 35 with 1x106 PFU of RSV A2. (A) Serum neutralization titers against RSV A2 were measured prior to challenge at day 35. Data is presented as the log2 dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. At sacrifice (day 39), (B) RSV F specific total IgG and (C) IgG1/IgG2a responses were measured in serum. Data bars represent the mean of 5 animals ± SD. *, P

PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine

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