Single Dose of a Polyanhydride Particle-Based Vaccine Generates Potent Antigen-Specific Antitumor Immune Responses.

Many factors affect vaccine efficacy. One of the most salient is the frequency and intervals of vaccine administration. In this study, we assessed the vaccine administration modality for a recently reported polyanhydride-based vaccine formulation, shown to generate antitumor activity. Polyanhydride particles encapsulating ovalbumin (OVA) were prepared using a double-emulsion technique and subcutaneously delivered to mice either as a single-dose or as prime-boost vaccine regimens in which two different time intervals between prime and boost were assessed (7 or 21 days). This was followed by measurement of cellular and humoral immune responses, and subsequent challenge of the mice with a lethal dose of E.G7-OVA cells to evaluate tumor protection. Interestingly, a single dose of the polyanhydride particle-based formulation induced sustained OVA-specific cellular immune responses just as effectively as the prime-boost regimens. In addition, mice receiving single-dose vaccine had similar levels of protection against tumor challenge compared with mice administered prime-boosts. In contrast, measurements of OVA-specific IgG antibody titers indicated that a booster dose was required to stimulate strong humoral immune responses, since it was observed that mice administered a prime-boost vaccine had significantly higher OVA-specific IgG1 serum titers than mice administered a single dose. These findings indicate that the requirement for a booster dose using these particles appears unnecessary for the generation of effective cellular immunity.

Pentaerythritol-based lipid A bolsters the antitumor efficacy of a polyanhydride particle-based cancer vaccine.

The primary objective of this study was to enhance the antitumor efficacy of a model cancer vaccine through co-delivery of pentaerythritol lipid A (PELA), an immunological adjuvant, and a model tumor antigen, ovalbumin (OVA), separately loaded into polyanhydride particles (PA). In vitro experiments showed that encapsulation of PELA into PA (PA-PELA) significantly enhanced its stimulatory capacity on dendritic cells as evidenced by increased levels of the cell surface costimulatory molecules, CD80/CD86. In vivo experiments showed that PA-PELA, in combination with OVA-loaded PA (PA-OVA), significantly expanded the OVA-specific CD8+ T lymphocyte population compared to PA-OVA alone. Furthermore, OVA-specific serum antibody titers of mice vaccinated with PA-OVA/PA-PELA displayed a significantly stronger shift toward a Th1-biased immune response compared to PA-OVA alone, as evidenced by the substantially higher IgG2C:IgG1 ratios achieved by the former. Analysis of E.G7-OVA tumor growth curves showed that mice vaccinated with PA-OVA/PA-PELA had the slowest average tumor growth rate.

Rational Design of Targeted Next-Generation Carriers for Drug and Vaccine Delivery.

Pattern recognition receptors on innate immune cells play an important role in guiding how cells interact with the rest of the organism and in determining the direction of the downstream immune response. Recent advances have elucidated the structure and function of these receptors, providing new opportunities for developing targeted drugs and vaccines to treat infections, cancers, and neurological disorders. C-type lectin receptors, Toll-like receptors, and folate receptors have attracted interest for their ability to endocytose their ligands or initiate signaling pathways that influence the immune response. Several novel technologies are being developed to engage these receptors, including recombinant antibodies, adoptive immunotherapy, and chemically modified antigens and drug delivery vehicles. These active targeting technologies will help address current challenges facing drug and vaccine delivery and lead to new tools to treat human diseases.

Automated High-Throughput Synthesis of Protein-Loaded Polyanhydride Nanoparticle Libraries.

The development of high-throughput techniques and combinatorial libraries can facilitate rapid synthesis and screening of biomaterial-based nanocarriers for drug and vaccine delivery. This study describes a high-throughput method using an automated robot for synthesizing polyanhydride nanoparticles encapsulating proteins. Polyanhydrides are a class of safe and biodegradable polymers that have been widely used as drug and vaccine delivery vehicles. The robot contains a multiplexed homogenizer and has the capacity to handle parallel streams of monomer or polymer solutions to synthesize polymers and/or nanoparticles. Copolymer libraries were synthesized using the monomers sebacic acid, 1,6-bis( p-carboxyphenoxy)hexane, and 1,8-bis( p-carboxyphenoxy)-3,6-dioxactane and compared to conventionally synthesized copolymers. Nanoparticle libraries of varying copolymer compositions encapsulating the model antigen ovalbumin were synthesized using flash nanoprecipitation. The amount of the surfactant Span 80 was varied to test its effect on protein encapsulation efficiency as well as antigen release kinetics. It was observed that, although the amount of surfactant did not significantly affect protein release rate, its presence enhanced protein encapsulation efficiency. Protein burst and release kinetics from conventionally and combinatorially synthesized nanoparticles were similar even though particles synthesized using the high-throughput technique were smaller. Finally, it was demonstrated that the high-throughput method could be adapted to functionalize the surface of particle libraries to aid in the design and screening of targeted drug and vaccine delivery systems. These results suggest that the new high-throughput method is a viable alternative to conventional methods for synthesizing and screening protein and vaccine delivery vehicles.

Polyanhydride Nanovaccine Induces Robust Pulmonary B and T Cell Immunity and Confers Protection Against Homologous and Heterologous Influenza A Virus Infections.

Influenza A virus (IAV) is a major cause of respiratory illness. Given the disease severity, associated economic costs, and recent appearance of novel IAV strains, there is a renewed interest in developing novel and efficacious "universal" IAV vaccination strategies. Recent studies have highlighted that immunizations capable of generating local (i.e., nasal mucosa and lung) tissue-resident memory T and B cells in addition to systemic immunity offer the greatest protection against future IAV encounters. Current IAV vaccines are designed to largely stimulate IAV-specific antibodies, but do not generate the lung-resident memory T and B cells induced during IAV infections. Herein, we report on an intranasally administered biocompatible polyanhydride nanoparticle-based IAV vaccine (IAV-nanovax) capable of providing protection against subsequent homologous and heterologous IAV infections in both inbred and outbred populations. Our findings also demonstrate that vaccination with IAV-nanovax promotes the induction of germinal center B cells within the lungs, both systemic and lung local IAV-specific antibodies, and IAV-specific lung-resident memory CD4 and CD8 T cells. Altogether our findings show that an intranasally administered nanovaccine can induce immunity within the lungs, similar to what occurs during IAV infections, and thus could prove useful as a strategy for providing "universal" protection against IAV.

The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response.

The goal of this research was to study the effect of polyanhydride chemistry on the immune response induced by a prophylactic cancer vaccine based on biodegradable polyanhydride particles. To achieve this goal, different compositions of polyanhydride copolymers based on 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), 1,6-bis-(p-carboxyphenoxy)-hexane (CPH), and sebacic anhydride (SA) were synthesized by melt polycondensation, and polyanhydride copolymer particles encapsulating a model antigen, ovalbumin (OVA), were then synthesized using a double emulsion solvent evaporation technique. The ability of three different compositions of polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. In addition, the impact of unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunological adjuvant, on the immune response was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50µg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25µg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8+ T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and provided longer protection against tumor challenge with an OVA-expressing thymoma cell line in comparison to formulations made from other polyanhydride copolymers. The results also revealed that vaccination with CpG ODN along with polyanhydride particles encapsulating OVA did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and provide important insights on rationally designing efficacious cancer vaccines. STATEMENT OF SIGNIFICANCE: Compared to soluble cancer vaccine formulations, tumor antigens encapsulated in biodegradable polymeric particles have been shown to sustain antigen release and provide long-term protection against tumor challenge by improving the immune response towards the antigen. Treatment of mice with cancer vaccines based on different polyanhydride copolymers encapsulating OVA resulted in stimulation of tumor-specific immune responses with different magnitudes. This clearly indicates that polyanhydride chemistry plays a substantial role in stimulating the immune response. Vaccination with 20:80 CPTEG:CPH/OVA, the most hydrophobic formulation, stimulated the strongest cellular and humoral immune responses and provided the longest survival outcome without adding any other adjuvant. The most important finding in this study is that the copolymer composition of polyanhydride particle-based vaccines can have a direct effect on the magnitude of the antitumor immune response and should be selected carefully in order to achieve optimal cancer vaccine efficacy.

Safety and biocompatibility of carbohydrate-functionalized polyanhydride nanoparticles.

Carbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-D-mannopyranosyl-(1,2)-D-mannopyranoside), galactose (specifically carboxymethyl-ß-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.