Alexander Marin

Background: The quest for well-defined immunoadjuvants remains one of the highest priorities for the successful development of effective vaccines. Combination adjuvants, which are designed to integrate both the ability to activate a variety of immune mechanisms and synergistically improve the delivery of vaccine components, are well-positioned to address the unmet needs. The development of a preventive vaccine against hepatitis C virus (HCV)—a major public health concern—is a particular instance in which the choice of the immunoadjuvant is of utmost importance. Methods: We assembled a lipid A Toll-like receptor 4 (TLR4) agonist BECC438 and TLR7/8 agonist resiquimod (R848) on a polyphosphazene macromolecule (PCPP) to create a nanoscale immunoadjuvant-vaccine delivery system: PCPP-R+BECC438. This aqueous-based system was formulated with the HCV sE2 antigen, and the resulting vaccine candidate was evaluated in vivo for the ability to induce immune responses. Results: Co-assembly of adjuvants resulted in a visually clear aqueous system of nanoscale dimensions, monomodal size distribution, and entropy-driven interactions between components. Intramuscular immunization of mice with HCV sE2 antigen formulated in a polyphosphazene-based nano-system induced ten-fold higher IgG and IgG2a titers than the antigen adjuvanted with BECC438 alone. PCPP-R+BECC438 formulated HCV sE2 also produced statistically significant improvements in IgG2a/IgG1 ratio and more robust HCVpp neutralization ID₅₀ titers than control formulations. Conclusions: Polyphosphazene-assembled adjuvant nano-system promotes in vivo immune responses of enhanced quantity and quality of antibodies with increased potency of HCV neutralization.

Background: The quest for well-defined immunoadjuvants remains one of the highest priorities for the successful development of effective vaccines. Combination adjuvants, which are designed to integrate both the ability to activate a variety of immune mechanisms and synergistically improve delivery of vaccine components, are well-positioned to address the unmet needs. The development of preventive vaccine against hepatitis C virus (HCV) – a major public health concern, is a particular instance in which choice of the immunoadjuvant is of utmost importance. Methods: We assembled a lipid A Toll-like receptor 4 (TLR4) agonist BECC438 and TLR7/8 agonist resiquimod (R848) on a polyphosphazene macromolecule (PCPP) to create a nanoscale immunoadjuvant-vaccine delivery system: PCPP-R+BECC438. This aqueous-based system was formulated with HCV sE2 antigen, and the resulting vaccine candidate was evaluated in vivo for the ability to induce immune responses. Results: Co-assembly of adjuvants resulted in a visually clear aqueous system of nanoscale dimensions, monomodal size distribution and entropy driven interactions between components. Intramuscular immunization of mice with HCV sE2 antigen formulated in polyphosphazene-based nano-system induced ten-fold higher IgG and IgG2a titers than the antigen adjuvanted with BECC438 alone. PCPP-R+BECC438 formulated HCV sE2 also produced statistically significant improvements in IgG2a/IgG1 ratio and more robust HCVpp neutralization ID50 titers than control formulations. Conclusions: Polyphosphazene-assembled adjuvant nano-system promotes in vivo immune responses of enhanced quantity and quality of antibodies with increased potency of HCV neutralization.

Hepatitis C virus (HCV) is a major public health concern, and the development of an effective HCV vaccine plays an important role in the effort to prevent new infections. Supramolecular co-assembly and co-presentation of the HCV envelope E1E2 heterodimer complex and core protein presents an attractive vaccine design strategy for achieving effective humoral and cellular immunity. With this objective, the two antigens were non-covalently assembled with an immunostimulant (TLR 7/8 agonist) into virus-mimicking polymer nanocomplexes (VMPNs) using a biodegradable synthetic polyphosphazene delivery vehicle. The resulting assemblies were characterized using dynamic light scattering and asymmetric flow field-flow fractionation methods and directly visualized in their vitrified state by cryogenic electron microscopy. The in vivo superiority of VMPNs over the individual components and an Alum-formulated vaccine manifests in higher neutralizing antibody titers, the promotion of a balanced IgG response, and the induction of a cellular immunity—CD4+ T cell responses to core proteins. The aqueous-based spontaneous co-assembly of antigens and immunopotentiating molecules enabled by a synthetic biodegradable carrier offers a simple and effective pathway to the development of polymer-based supramolecular nanovaccine systems.

Hepatitis C virus (HCV) is a major public health concern, and the development of an effective HCV vaccine plays an important role in an effort to prevent new infections. Supramolecular co-assembly and co-presentation of the HCV envelope E1E2 heterodimer complex and core protein presents an attractive vaccine design strategy for achieving effective humoral and cellular immunity. With this objective, the two antigens were non-covalently assembled with a TLR 7/8 agonist into virus-mimicking polymer nanocomplexes (VMPNs) using biodegradable synthetic polyphosphazene delivery vehicle. The resulting assemblies were characterized using dynamic light scattering and asymmetric flow field flow fractionation methods and directly visualized in their vitrified state by cryogenic electron microscopy. The in vivo superiority of VMPNs over the individual components and an Alum formulated vaccine manifests in higher neutralizing antibody titers, promotion of a balanced IgG response, and induction of CD4+ T cell responses to core proteins. Aqueous-based spontaneous co-assembly of antigens and immunopotentiating molecules enabled by a synthetic biodegradable carrier offers a simple and effective pathway to the development of polymer-based supramolecular vaccine systems.

The inclusion of fluorine motifs in drugs and drug delivery systems is an established tool for modulating their biological potency. Fluorination can improve drug specificity or boost the vehicle’s ability to cross cellular membranes. However, the approach has yet to be applied to vaccine adjuvants. Herein, the synthesis of fluorinated bioisostere of a clinical stage immunoadjuvant—poly[di(carboxylatophenoxy)phosphazene], PCPP—is reported. The structure of water-soluble fluoropolymer—PCPP-F, which contains two fluorine atoms per repeat unit—was confirmed using ¹H, ³¹P and ¹⁹F NMR, and its molecular mass and molecular dimensions were determined using size-exclusion chromatography and dynamic light scattering. Insertion of fluorine atoms in the polymer side group resulted in an improved solubility in acidic solutions and faster hydrolytic degradation rate, while the ability to self-assemble with an antigenic protein, lysozyme—an important feature of polyphosphazene vaccine adjuvants—was preserved. In vivo assessment of PCPP-F demonstrated its greater ability to induce antibody responses to Hepatitis C virus antigen when compared to its non-fluorinated counterpart. Taken together, the superior immunoadjuvant activity of PCPP-F, along with its improved formulation characteristics, demonstrate advantages of the fluorination approach for the development of this family of macromolecular vaccine adjuvants.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant—PCPP and a model antigen—lysozyme in a physiologically relevant environment—saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance (wNMR), and dynamic light scattering (DLS). The formation of lysozyme–PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of wNMR method for studying protein-polymer interactions, especially for low protein-load complexes.

Self-assembly of ionically charged small molecule drugs with water-soluble biodegradable polyelectrolytes into nano-scale complexes can potentially offer a novel and attractive approach to improving drug solubility and prolonging its half-life. Nanoassemblies of quisinostat with water-soluble PEGylated anionic polyphosphazene were prepared by gradient-driven escape of solvent resulting in the reduction of solvent quality for a small molecule drug. A study of binding, analysis of composition, stability, and release profiles was conducted using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) spectroscopy. Potency assays were performed with WM115 human melanoma and A549 human lung cancer cell lines. The resulting nano-complexes contained up to 100 drug molecules per macromolecular chain and displayed excellent water-solubility and improved hemocompatibility when compared to co-solvent-based drug formulations. Quisinostat release time (complex dissociation) at near physiological conditions in vitro varied from 5 to 14 days depending on initial drug loading. Multimeric complexes displayed dose-dependent potency in cell-based assays and the results were analyzed as a function of complex concentration, as well as total content of drug in the system. The proposed self-assembly process may present a simple alternative to more sophisticated delivery modalities, namely chemically conjugated prodrug systems and nanoencapsulation-based formulations.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.