Matthew D. Gallovic

Rapid vaccination using an acetalated dextran microparticulate subunit vaccine confers protection against triplicate challenge by bacillus anthracis.

PurposeA rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis.MethodWe formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist).ResultsWe were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis.ConclusionsResults indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

Delivery of host cell-directed therapeutics for intracellular pathogen clearance.

Intracellular pathogens present a major health risk because of their innate ability to evade clearance. Their location within host cells and ability to react to the host environment by mutation or transcriptional changes often enables survival mechanisms to resist standard therapies. Host-directed drugs do not target the pathogen, minimizing the potential development of drug resistance; however, they can be difficult to deliver efficiently to intracellular sites. Vehicle delivery of host-mediated response drugs not only improves drug distribution and toxicity profiles, but can reduce the total amount of drug necessary to clear infection. In this article, we will review some host-directed drugs and current drug delivery techniques that can be used to efficiently clear intracellular infections.

Acetalated Dextran Microparticulate Vaccine Formulated via Coaxial Electrospray Preserves Toxin Neutralization and Enhances Murine Survival Following Inhalational Bacillus Anthracis Exposure

Subunit formulations are regarded as the safest type of vaccine, but they often contain a protein-based antigen that can result in significant challenges, such as preserving antigenicity during formulation and administration. Many studies have demonstrated that encapsulation of protein antigens in polymeric microparticles (MPs) via emulsion techniques results in total IgG antibody titers comparable to alum formulations, however, the antibodies themselves are non-neutralizing. To address this issue, a coaxial electrohydrodynamic spraying (electrospray) technique is used to formulate a microparticulate-based subunit anthrax vaccine under conditions that minimize recombinant protective antigen (rPA) exposure to harsh solvents and high shear stress. rPA and the adjuvant resiquimod are encapsulated either in separate or the same acetalated dextran MPs. Using a murine model, the electrospray formulations lead to higher IgG2a subtype titers as well as comparable total IgG antibody titers and toxin neutralization relative to the FDA-approved vaccine (BioThrax). BioThrax provides no protection against a lethal inhalational challenge of the highly virulent Ames Bacillus anthracis anthrax strain, whereas 50% of the mice vaccinated with separately encapsulated electrospray MPs survive. Overall, this study demonstrates the potential use of electrospray for encapsulating protein antigens in polymeric MPs.

Evaluation of a biodegradable microparticulate polymer as a carrier for Burkholderia pseudomallei subunit vaccines in a mouse model of melioidosis.

Melioidosis, a potentially lethal disease of humans and animals, is caused by the soil-dwelling bacterium Burkholderia pseudomallei. Due to B. pseudomalleis classification as a Tier 1 Select Agent, there is substantial interest in the development of an effective vaccine. Yet, despite decades of research, no effective target, adjuvant or delivery vehicle capable of inducing protective immunity against B. pseudomallei infection has been identified. We propose a microparticulate delivery vehicle comprised of the novel polymer acetalated dextran (Ac-DEX). Ac-DEX is an acid-sensitive biodegradable carrier that can be fabricated into microparticles (MPs) that are relatively stable at pH 7.4, but rapidly degrade after phagocytosis by antigen presenting cells where the pH can drop to 5.0. As compared to other biomaterials, this acid sensitivity has been shown to enhance cross presentation of subunit antigens. To evaluate this platform as a delivery system for a melioidosis vaccine, BALB/c mice were vaccinated with Ac-DEX MPs separately encapsulating B. pseudomallei whole cell lysate and the toll-like receptor (TLR) 7/8 agonist resiquimod. This vaccine elicited a robust antibody response that included both Th1 and Th2 immunity. Following lethal intraperitoneal challenge with B. pseudomallei 1026b, vaccinated mice demonstrated a significant delay to time of death compared to untreated mice. The formulation, however, demonstrated incomplete protection indicating that lysate protein offers limited value as an antigen. Nevertheless, our Ac-DEX MPs may offer an effective delivery vehicle for a subunit B. psuedomallei vaccine.

Electrospun Acetalated Dextran Scaffolds for Temporal Release of Therapeutics

Electrospun acetalated dextran (Ac-DEX) scaffolds were fabricated to encapsulate resiquimod, an immunomodulatory toll-like-receptor (TLR) agonist. Ac-DEX has been used to fabricate scaffolds for sustained and temporal delivery of therapeutics because it has tunable degradation rates that are dependent on its synthesis reaction time or the molecular weight of dextran. Additionally, as opposed to commonly electrospun polyesters that shift the local pH upon degradation, the degradation products of Ac-DEX are pH-neutral: dextran, an alcohol, and the metabolic byproduct acetone. Formulations of Ac-DEX with two different degradation rates were used in this study. The effects of electrospinning conditions on the scaffold size and morphology were examined as well as fibroblast adhesion as imaged with fluorescence microcopy and scanning electron microscopy. Macrophage (MΦ) viability further indicates that the scaffolds are cytocompatible. Also, the controlled release profiles of resiquimod from loaded scaffolds and nitric oxide (NO) production by MΦ incubated with these scaffolds show the potential for Ac-DEX scaffolds to be used to temporally and efficiently deliver therapeutics. Overall, we present a novel scaffold that can have tunable and unique drug release rates for tissue engineering, drug delivery, immunomodulation, and wound healing applications.

Saquinavir Loaded Acetalated Dextran Microconfetti – a Long Acting Protease Inhibitor Injectable

PurposeSince the adoption of highly active antiretroviral therapy, HIV disease progression has slowed across the world; however, patients are often required to take multiple medications daily of poorly bioavailable drugs via the oral route, leading to gastrointestinal irritation. Recently, long acting antiretroviral injectables that deliver drug for months at a time have moved into late phase clinical trials. Unfortunately, these solid phase crystal formulations have inherent drawbacks in potential dose dumping and a greater likelihood for burst release of drug compared to polymeric formulations.MethodsUsing electrospinning, acetalated dextran scaffolds containing the protease inhibitor saquinavir were created. Grinding techniques were then used to process these scaffolds into injectables which are termed saquinavir microconfetti. Microconfetti was analyzed for in vitro and in vivo release kinetics.ResultsHighly saquinavir loaded acetalated dextran electrospun fibers were able to be formed and processed into saquinavir microconfetti while other polymers such as poly lactic-co-glycolic acid and polycaprolactone were unable to do so. Saquinavir microconfetti release kinetics were able to be tuned via drug loading and polymer degradation rates. In vivo, a single subcutaneous injection of saquinavir microconfetti released drug for greater than a week with large tissue retention.ConclusionsMicroconfetti is a uniquely tunable long acting injectable that would reduce the formation of adherence related HIV resistance. Our findings suggest that the injectable microconfetti delivery system could be used for long acting controlled release of saquinavir and other hydrophobic small molecule drugs.

A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination

Abstract Most FDA‐approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly‐understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)‐driven anti‐viral immunity; however, a major hindrance is STINGs cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3′3′‐cGAMP (cGAMP), encapsulated in acid‐sensitive acetalated dextran (Ace‐DEX) polymeric microparticles (MPs) which passively target antigen‐presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace‐DEX cGAMP MPs enhanced type‐I interferon responses nearly 1000‐fold in vitro and 50‐fold in vivo, caused up to a 104‐fold boost in antibody titers, increased Th1‐associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post‐immunization when using CDN adjuvant doses up to 100‐fold lower than previous reports. For these reasons, Ace‐DEX MP‐encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity. Graphical abstract Figure. No Caption available.

Tunable degradation of acetalated dextran microparticles enables controlled vaccine adjuvant and antigen delivery to modulate adaptive immune responses

&NA; Subunit vaccines are often poorly immunogenic, and adjuvants and/or delivery vehicles, such as polymeric microparticles (MPs), can be used to enhance immune responses. MPs can also be used to understand cell activation kinetics and the significant impact antigen and adjuvant release has on adaptive immune responses. By controlling antigen and adjuvant release, we can determine if it is important to have precise temporal control over release of these elements to optimize the peak and duration of protective immunity and improve vaccine safety profiles. In order to study the effect of tunable adjuvant or antigen delivery on generation of adaptive immunity, we used acetalated dextran (Ace‐DEX) MPs. Ace‐DEX MPs were used because their tunable degradation can be controlled based on polymer cyclic acetal coverage (CAC). Ace‐DEX MPs of varying degradation profiles were used to deliver murabutide or ovalbumin (OVA) as a model adjuvant or antigen, respectively. When murabutide was encapsulated within Ace‐DEX MPs to test for controlled adjuvant delivery, fast‐degrading MPs exhibited higher humoral and cellular responses in vivo at earlier time points, while slow‐degrading MPs resulted in stronger responses at later time points. When OVA was encapsulated within Ace‐DEX MPs to test for controlled antigen delivery, fast‐degrading MPs induced greater antibody and cytokine production throughout the length of the experiment. This differential response suggests the need for distinct, flexible control over adjuvant or antigen delivery and its impact on immune response modulation.

Microparticles formulated from a family of novel silylated polysaccharides demonstrate inherent immunostimulatory properties and tunable hydrolytic degradability

Acid-degradable polymers are well-suited for use as drug delivery vehicles because numerous physiological sites (e.g., intracellular endocytic pathway) are acidic. Here we report the synthesis of acid-sensitive silylated polysaccharides derived from either dextran or inulin with various alkyl substitutions on the silicon center: trimethylsilyl dextran (TMS-DEX), ethyldimethylsilyl dextran (EDMS-DEX), triethylsilyl dextran (TES-DEX), and trimethylsilyl inulin (TMS-IN). The silylated dextran (Silyl-DEX) and silylated inulin (Silyl-IN) polymers were fabricated into microparticles (MPs) via emulsification followed by solvent evaporation. These MPs were relatively stable at extracellular pH 7.4 and displayed a wide range of pH 2.0 and 5.0 degradation half-lives (fifteen minutes to greater than nine days) that were dependent on the extent of silylation (40 to 98%) and steric crowding on the silicon center (trimethyl to ethyldimethyl to triethyl). Silyl-DEX and Silyl-IN MPs exhibited cytocompatibility when cultured in vitro with RAW 264.7 macrophages. TES-DEX and TMS-IN MPs, composed of highly hydrophobic moieties and the parent immunostimulatory inulin, respectively, elicited substantial in vitro production of tumor necrosis factor alpha, a cytokine associated with an innate immune response. In vivo immunization with a model ovalbumin antigen encapsulated in silylated polysaccharide MPs, without a separate adjuvant, resulted in a dual humoral and cellular response that was superior to an alum-adjuvanted formulation. Overall, we present Silyl-DEX and Silyl-IN as members of the acid-degradable polymer family for potential use in subunit vaccines and other drug delivery applications.

In Vivo and Cellular Trafficking of Acetalated Dextran Microparticles for Delivery of a Host-Directed Therapy for Salmonella enterica Serovar Typhi Infection

Previously we have encapsulated host-directed therapy AR-12 into acetalated dextran (Ace-DEX) microparticles (MPs) to mitigate drug toxicity and passively target phagocytic host cells. Herein, we have improved upon our initial emulsion-based formulation of Ace-DEX MPs encapsulating AR-12 (AR-12/MPs) by improving the drug encapsulation efficiency, evaluating sterilization processes for manufacturing, and understanding cellular and in vivo trafficking of the MPs. By using an alternative solvent system, ethyl acetate, we report an increased encapsulation efficiency of AR-12 while maintaining the pH-responsive degradation kinetics of Ace-DEX MPs. To better manufacture this novel antimicrobial formulation, we sterilized AR-12/MPs by gamma irradiation or ethylene oxide and evaluated their efficacy against intracellular Salmonella enterica serovar Typhi. Sterilized AR-12/MPs resulted in a significant reduction in intracellular bacterial burden compared to Blank/MPs. We also characterized intracellular trafficking of Ace-DEX MPs encapsulating fluorophores, which demonstrated internalization of MPs in endo/lysosomal compartments and time and degradation-rate dependent lysosomal escape into cytosolic compartments. Additionally, in vivo toxicity was mitigated following encapsulation of AR-12, where the maximum tolerated dose of AR-12 was increased compared to soluble treatment via intranasal, intravenous, and intraperitoneal administration routes. Following in vivo trafficking of Ace-DEX MPs via the same routes, intranasal administration demonstrated the highest accumulation in the lungs, liver, and kidneys, which persisted out to 240 h. Overall, we have advanced the formulation of this host-directed therapy and broadened the understanding of Ace-DEX MP delivery.