Kathleen A. Ross

Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery.

Efficacy, purity, safety, and potency are important attributes of vaccines. Polyanhydride particles represent a novel class of vaccine adjuvants and delivery platforms that have demonstrated the ability to enhance the stability of protein antigens as well as elicit protective immunity against bacterial pathogens. This work aims to elucidate the biocompatibility, inflammatory reactions, and particle effects on mice injected with a 5 mg dose of polyanhydride nanoparticles via common parenteral routes (subcutaneous and intramuscular). Independent of polymer chemistry, nanoparticles more effectively disseminated away from the injection site as compared to microparticles, which exhibited a depot effect. Using fluorescent probes, the in vivo distribution of three formulations of nanoparticles, following subcutaneous administration, indicated migration away from the injection site. Less inflammation was observed at the injection sites of mice-administered nanoparticles as compared to Alum and incomplete Freunds adjuvant. Furthermore, histological evaluation revealed minimal adverse injection site reactions and minimal toxicological effects associated with the administration of nanoparticles at 30 days post-administration. Collectively, these results demonstrate that polyanhydride nanoparticles do not induce inflammation as a cumulative effect of particle persistence or degradation and are, therefore, a viable candidate for a vaccine delivery platform.

Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses

Microparticle adjuvants based on biodegradable polyanhydrides were used to provide controlled delivery of a model antigen, ovalbumin (Ova), to mice. Ova was encapsulated into two different polyanhydride microparticle formulations to evaluate the influence of polymer chemistry on the nature and magnitude of the humoral immune response after administration of a suboptimal dose. Subcutaneous administration of a single dose of polyanhydride microparticles containing 25 μg of Ova elicited humoral immune responses that were comparable in magnitude to that induced by soluble doses of 400-1600 μg Ova. In contrast, the avidity of the Ova-specific antibodies was greater in mice administered the microparticle formulations in comparison to the higher soluble doses. Finally, the microparticle delivery system primed an anamnestic immune response as evidenced by the significant increases in Ova-specific antibody when mice were administered an antigenic challenge of 25 μg of Ova at 12 weeks post-vaccination. Together, these results indicate that encapsulation of antigens into polyanhydride microparticles facilitates isotype switching, establishes immunologic memory, and the humoral response was characterized by a higher quality antibody response.

Structural and antigenic stability of H5N1 hemagglutinin trimer upon release from polyanhydride nanoparticles

Although H5N1 avian influenza has not yet acquired the capacity to readily infect humans, should it do so, this viral pathogen would present an increasing threat to the immunologically naïve human population. Subunit vaccines based on the viral glycoprotein hemagglutinin (HA) can provide protective immunity against influenza. Polyanhydride nanoparticles have been shown to enhance efficacy of subunit vaccines, providing the dual advantages of adjuvanticity and sustained delivery resulting in enhanced protein stability and immunogenicity. In this work, a recombinant trimer of H5 (H53 ) was encapsulated and released from polyanhydride nanoparticles. Release kinetics of the encapsulated H53 were found to be dependent on polymer chemistry (i.e., hydrophobicity and molecular weight). Polyanhydride nanoparticles composed of sebacic anhydride and 1,6-bis(p-carboxyphenoxy)hexane (CPH; that degrade into more acidic monomers) released structurally stable HA H53 , while H53 released from formulations composed of CPH and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) (that are amphiphilic and whose degradation products are less acidic) displayed unfolding of tertiary structure. However, the antigenicity of the H53 based on binding of a H5-specific monoclonal antibody was preserved upon release from all the formulations studied, demonstrating the value of polyanhydride nanoparticles as a viable platform for HA-based influenza vaccines.

Lung Deposition and Cellular Uptake Behavior of Pathogen‐Mimicking Nanovaccines in the First 48 Hours

Pulmonary immunization poses the unique challenge of balancing vaccine efficacy with minimizing inflammation in the respiratory tract. While previous studies have shown that mice immunized intranasally with F1-V-loaded polyanhydride nanoparticles are protected from a lethal challenge with Yersinia pestis, little is known about the initial interaction between the nanoparticles and immune cells following intranasal administration. Here, the deposition within the lung and internalization by phagocytic cells of polyanhydride nanovaccines encapsulating F1-V are compared with that of soluble F1-V alone or F1-V adjuvanted with monophosphoryl lipid A (MPLA). Encapsulation of F1-V into polyanhydride nanoparticles prolonged its presence while F1-V administered with MPLA is undetectable within 48 h. The inflammation induced by the polyanhydride nanovaccine is mild compared with the marked inflammation induced by the MPLA-adjuvanted F1-V. Even though F1-V delivered with saline is detected in the lung 48 h after administration, it is known that this regimen does not elicit a protective immune response. The prolonged F1-V presence in the lung in concert with the mild inflammatory response provided by the nanovaccine provides new insights into the development of protective immune responses with a single intranasal dose.

Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios.

Nano-enabled delivery of diverse payloads across complex biological barriers

Complex biological barriers are major obstacles for preventing and treating disease. Nanocarriers are designed to overcome such obstacles by enhancing drug delivery through physiochemical barriers and improving therapeutic indices. This review critically examines both biological barriers and nanocarrier payloads for a variety of drug delivery applications. A spectrum of nanocarriers is discussed that have been successfully developed for improving tissue penetration for preventing or treating a range of infectious, inflammatory, and degenerative diseases.

Polyanhydride nanovaccine platform enhances antigen-specific cytotoxic T cell responses

Polyanhydride nanoparticle-based vaccines (or nanovaccines) stabilize protein antigens, provide sustained antigen release leading to prolonged antigen presence, enhance activation of antigen presenting cells, and elicit protective immunity against respiratory infections upon challenge. However, induction of cell-mediated immunity when mice are immunized with polyanhydride nanovaccines has not been evaluated. Using a transgenic ovalbumin-specific T cell adoptive transfer model, we report the induction of antigen-specific cytotoxic CD8+ T cells expressing an effector memory phenotype by seven days after immunization with nanovaccine formulations. Furthermore, mice immunized with polyanhydride nanovaccines demonstrated enhanced recall responses after antigen re-exposure 35 days post-immunization indicating the activation and recruitment of antigen-specific memory CD8+ T cells to the site of antigen deposition.

pH-Responsive Microencapsulation Systems for the Oral Delivery of Polyanhydride Nanoparticles

Multicompartmental polymer carriers, referred to as Polyanhydride-Releasing Oral MicroParticle Technology (PROMPT), were formed by a pH-triggered antisolvent precipitation technique. Polyanhydride nanoparticles were encapsulated into anionic pH-responsive microparticle gels, allowing for nanoparticle encapsulation in acidic conditions and subsequent release in neutral pH conditions. The effects of varying the nanoparticle composition and feed ratio on the encapsulation efficiency were evaluated. Nanoparticle encapsulation was confirmed by confocal microscopy and infrared spectroscopy. pH-triggered protein delivery from PROMPT was explored using ovalbumin (ova) as a model drug. PROMPT microgels released ova in a pH-controlled manner. Increasing the feed ratio of nanoparticles into the microgels increased the total amount of ova delivered, as well as decreased the observed burst release. The cytocompatibility of the polymer materials were assessed using cells representative of the GI tract. Overall, these results suggest that pH-dependent microencapsulation is a viable platform to achieve targeted intestinal delivery of polyanhydride nanoparticles and their payload(s).

Emerging trends in the immunotherapy of pancreatic cancer

Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the U.S., claiming approximately 43,000 lives every year. Much like other solid tumors, PC evades the host immune surveillance by manipulating immune cells to establish an immunosuppressive tumor microenvironment (TME). Therefore, targeting and reinstating the patients immune system could serve as a powerful therapeutic tool. Indeed, immunotherapy has emerged in recent years as a potential adjunct treatment for solid tumors including PC. Immunotherapy modulates the hosts immune response to tumor-associated antigens (TAAs), eradicates cancer cells by reducing host tolerance to TAAs and provides both short- and long-term protection against the disease. Passive immunotherapies like monoclonal antibodies or engineered T-cell based therapies directly target tumor cells by recognizing TAAs. Active immunotherapies, like cancer vaccines, on the other hand elicit a long-lasting immune response via activation of the patients immune cells against cancer cells. Several immunotherapy strategies have been tested for anti-tumor responses alone and in combination with standard care in multiple preclinical and clinical studies. In this review, we discuss various immunotherapy strategies used currently and their efficacy in abrogating self-antigen tolerance and immunosuppression, as well as their ability to eradicate PC.

Single dose of 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.