Polyanhydride Nanoparticles Research Articles

Bovine Respiratory Syncytial Virus Nanovaccine Induces Long-Lasting Humoral Immunity in Mice.

With limited therapies and vaccines available, human respiratory syncytial virus (HRSV) has a significant negative health impact on all age groups but particularly on infants, young children, and older adults. Bovine respiratory syncytial virus (BRSV) is pathogenically and antigenically similar to HRSV. Building upon previous studies using a BRSV nanovaccine coencapsulating multiple proteins, this work demonstrates the development and comparative evaluation of a coencapsulated nanovaccine to a cocktail nanovaccine formulation composed of polyanhydride nanoparticles encapsulating BRSV postfusion (F) glycoprotein and CpG ODN 1668 coadjuvant delivered simultaneously with nanoparticles encapsulating BRSV attachment glycoprotein (G) and CpG ODN 1668. These nanovaccine formulations were administered to C57BL/6 mice by one of two prime-boost regimens (i.e., intranasal/intranasal or intranasal/subcutaneous) followed by assessment of humoral immunity. The cocktail nanovaccine induced sustained anti-F and anti-G serum IgG antibody responses for 12 weeks postprimary immunization. Using polyanhydride particles to deliver G protein in a prime-boost regime also significantly induced serum anti-G antibodies compared to protein and coadjuvant alone. Serum IgG induced by the nanovaccine demonstrated virus-neutralizing capability from 42 to 119 days postprimary immunization. Further, anti-F IgG antibodies were detected in the bronchoalveolar lavage fluid of vaccinated animals. Finally, the nanovaccine induced long-lived anti-F antibody secreting plasma cells that were detectable in the bone marrow 205 days postprimary immunization. Overall, the BRSV nanovaccine(s) successfully induced long-lived humoral immune responses capable of virus neutralization, making this a promising vaccine candidate for further evaluation in other relevant animal models.

The NADPH Oxidase Inhibitor, Mitoapocynin, Mitigates DFP-Induced Reactive Astrogliosis in a Rat Model of Organophosphate Neurotoxicity.

NADPH oxidase (NOX) is a primary mediator of superoxides, which promote oxidative stress, neurodegeneration, and neuroinflammation after diisopropylfluorophosphate (DFP) intoxication. Although orally administered mitoapocynin (MPO, 10 mg/kg), a mitochondrial-targeted NOX inhibitor, reduced oxidative stress and proinflammatory cytokines in the periphery, its efficacy in the brain regions of DFP-exposed rats was limited. In this study, we encapsulated MPO in polyanhydride nanoparticles (NPs) based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and sebacic anhydride (SA) for enhanced drug delivery to the brain and compared with a high oral dose of MPO (30 mg/kg). NOX2 (GP91phox) regulation and microglial (IBA1) morphology were analyzed to determine the efficacy of MPO-NP vs. MPO-oral in an 8-day study in the rat DFP model. Compared to the control, DFP-exposed animals exhibited significant upregulation of NOX2 and a reduced length and number of microglial processes, indicative of reactive microglia. Neither MPO treatment attenuated the DFP effect. Neurodegeneration (FJB+NeuN) was significantly greater in DFP-exposed groups regardless of treatment. Interestingly, neuronal loss in DFP+MPO-treated animals was not significantly different from the control. MPO-oral rescued inhibitory neuronal loss in the CA1 region of the hippocampus. Notably, MPO-NP and MPO-oral significantly reduced astrogliosis (absolute GFAP counts) and reactive gliosis (C3+GFAP). An analysis of inwardly rectifying potassium channels (Kir4.1) in astroglia revealed a significant reduction in the brain regions of the DFP+VEH group, but MPO had no effect. Overall, both NP-encapsulated and orally administered MPO had similar effects. Our findings demonstrate that MPO effectively mitigates DFP-induced reactive astrogliosis in several key brain regions and protects neurons in CA1, which may have long-term beneficial effects on spontaneous seizures and behavioral comorbidities. Long-term telemetry and behavioral studies and a different dosing regimen of MPO are required to understand its therapeutic potential.

Aggregation-Inhibiting scFv-Based Therapies Protect Mice against AAV1/2-Induced A53T-α-Synuclein Overexpression.

To date, there is no cure for Parkinson's disease (PD). There is a pressing need for anti-neurodegenerative therapeutics that can slow or halt PD progression by targeting underlying disease mechanisms. Specifically, preventing the build-up of alpha-synuclein (αSyn) and its aggregated and mutated forms is a key therapeutic target. In this study, an adeno-associated viral vector loaded with the A53T gene mutation was used to induce rapid αSyn-associated PD pathogenesis in C57BL/6 mice. We tested the ability of a novel therapeutic, a single chain fragment variable (scFv) antibody with specificity only for pathologic forms of αSyn, to protect against αSyn-induced neurodegeneration, after unilateral viral vector injection in the substantia nigra. Additionally, polyanhydride nanoparticles, which provide sustained release of therapeutics with dose-sparing properties, were used as a delivery platform for the scFv. Through bi-weekly behavioral assessments and across multiple post-mortem immunochemical analyses, we found that the scFv-based therapies allowed the mice to recover motor activity and reduce overall αSyn expression in the substantia nigra. In summary, these novel scFv-based therapies, which are specific exclusively for pathological aggregates of αSyn, show early promise in blocking PD progression in a surrogate mouse PD model.

Nanotherapeutic delivery of antibiotic cocktail enhances intra-macrophage killing of Mycobacterium marinum

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in cold-blooded animals and is an opportunistic pathogen in humans. M. marinum is the closest genetic relative of the Mycobacterium tuberculosis complex and is a reliable surrogate for drug susceptibility testing. We synthesized and evaluated two nanoparticle (NP) formulations for compatibility with rifampicin, isoniazid, pyrazinamide, and ethambutol (PIRE), the front-line antimycobacterial drugs used in combination against active tuberculosis infections. Improved in vitro antimicrobial activity was observed with encapsulated rifampicin alone or in a cocktail of drugs formulated through co-encapsulation in amphiphilic polyanhydride NPs. Broth antimicrobial testing revealed that the encapsulation of PIRE in NP resulted in a significant increase in antimicrobial activity, with the benefit over soluble formulations at biologically relevant concentrations ranging from >10 to >3,000 fold. M. marinum-infected human macrophages treated with NP-PIRE were cleared of viable bacteria in 48 h following a single treatment, representing a >4 log reduction in colony-forming units and a >2,000-fold increase in antimicrobial activity. The amphiphilic polyanhydride nanoparticles demonstrated the ability to co-encapsulate PIRE antibiotics and enhance their antimicrobial activity against M. marinum in infected macrophages in culture and in vitro. These data suggest that polyanhydride nanoparticles are a promising nanotherapeutic for combatting Mycobacterium infections through improved intracellular targeting of encapsulated antibiotics.

Novel nanoadjuvants balance immune activation with modest inflammation: implications for older adult vaccines

BackgroundAge-associated impairments of immune response and inflammaging likely contribute to poor vaccine efficacy. An appropriate balance between activation of immune memory and inflammatory response may be more effective in vaccines for older adults; attempts to overcome reduced efficacy have included the addition of adjuvants or increased antigenic dose. Next generation vaccine formulations may also use biomaterials to both deliver and adjuvant vaccine antigens. In the context of aging, it is important to determine the degree to which new biomaterials may enhance antigen-presenting cell (APC) functions without inducing potent inflammatory responses of APCs or other immune cell types (e.g., T cells). However, the effect of newer biomaterials on these cell types from young and older adults remains unknown.ResultsIn this pilot study, cells from young and older adults were used to evaluate the effect of novel biomaterials such as polyanhydride nanoparticles (NP) and pentablock copolymer micelles (Mi) and cyclic dinucleotides (CDN; a STING agonist) on cytokine and chemokine secretion in comparison to standard immune activators such as lipopolysaccharide (LPS) and PMA/ionomycin. The NP treatment showed adjuvant-like activity with induction of inflammatory cytokines, growth factors, and select chemokines in peripheral blood mononuclear cells (PBMCs) of both young (n = 6) and older adults (n = 4), yet the degree of activation was generally less than LPS. Treatment with Mi or CDN resulted in minimal induction of cytokines and chemokine secretion with the exception of increased IFN-α and IL-12p70 by CDN. Age-related decreases were observed across multiple cytokines and chemokines, yet IFN-α, IL-12, and IL-7 production by NP or CDN stimulation was equal to or greater than in cells from younger adults. Consistent with these results in aged humans, a combination nanovaccine composed of NP, Mi, and CDN administered to aged mice resulted in a greater percentage of antigen-specific CD4+ T cells and greater effector memory cells in draining lymph nodes compared to an imiquimod-adjuvanted vaccine.ConclusionsOverall, our novel biomaterials demonstrated a modest induction of cytokine secretion with a minimal inflammatory profile. These findings suggest a unique role for biomaterial nanoadjuvants in the development of next generation vaccines for older adults.

“Just right” combinations of adjuvants with nanoscale carriers activate aged dendritic cells without overt inflammation

BackgroundThe loss in age-related immunological markers, known as immunosenescence, is caused by a combination of factors, one of which is inflammaging. Inflammaging is associated with the continuous basal generation of proinflammatory cytokines. Studies have demonstrated that inflammaging reduces the effectiveness of vaccines. Strategies aimed at modifying baseline inflammation are being developed to improve vaccination responses in older adults. Dendritic cells have attracted attention as an age-specific target because of their significance in immunization as antigen presenting cells that stimulate T lymphocytes.ResultsIn this study, bone marrow derived dendritic cells (BMDCs) were generated from aged mice and used to investigate the effects of combinations of adjuvants, including Toll-like receptor, NOD2, and STING agonists with polyanhydride nanoparticles and pentablock copolymer micelles under in vitro conditions. Cellular stimulation was characterized via expression of costimulatory molecules, T cell-activating cytokines, proinflammatory cytokines, and chemokines. Our results indicate that multiple TLR agonists substantially increase costimulatory molecule expression and cytokines associated with T cell activation and inflammation in culture. In contrast, NOD2 and STING agonists had only a moderate effect on BMDC activation, while nanoparticles and micelles had no effect by themselves. However, when nanoparticles and micelles were combined with a TLR9 agonist, a reduction in the production of proinflammatory cytokines was observed while maintaining increased production of T cell activating cytokines and enhancing cell surface marker expression. Additionally, combining nanoparticles and micelles with a STING agonist resulted in a synergistic impact on the upregulation of costimulatory molecules and an increase in cytokine secretion from BMDCs linked with T cell activation without excessive secretion of proinflammatory cytokines.ConclusionsThese studies provide new insights into rational adjuvant selection for vaccines for older adults. Combining appropriate adjuvants with nanoparticles and micelles may lead to balanced immune activation characterized by low inflammation, setting the stage for designing next generation vaccines that can induce mucosal immunity in older adults.

Long-Lasting Protection Induced by a Polyanhydride Nanovaccine against Respiratory Syncytial Virus in an Outbred Mouse Model.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children. In humans, natural infection with RSV affords only partial long-term protection from reinfection, and there is no licensed RSV vaccine currently available. We have developed a new vaccine candidate, termed RSVNanoVax, composed of polyanhydride nanoparticles encapsulating the RSV prefusion F protein and a CpG 1668 oligodeoxynucleotide adjuvant. We recently reported that vaccination of inbred BALB/c mice with RSVNanoVax induced both RSV-specific cellular and humoral immunity, which provided protection from viral replication and RSV-induced disease. To further assess the efficacy of RSVNanoVax, here, we utilized outbred Swiss Webster mice to examine vaccine efficacy in a more genetically diverse population. Following intranasal prime-boost vaccination with RSVNanoVax, Swiss Webster mice exhibited robust titers of systemic RSV F-directed IgG antibodies and RSV F-directed IgA within the lungs and nasal passages that were sustained out to at least 1 year post-vaccination. Serum antibodies maintained robust neutralizing activity against both RSV A and B strains. Following RSV challenge, vaccinated Swiss Webster mice exhibited rapid viral clearance from the lungs. Overall, our results indicate that RSVNanoVax represents a promising RSV vaccine candidate capable of providing long-term protection and immunity in a genetically diverse population. IMPORTANCE Respiratory syncytial virus (RSV) infection causes thousands of infections and deaths in children and elderly adults each year. Research in this field is of great importance as there remains no licensed vaccine to prevent RSV infections. We developed a novel vaccine candidate, RSVNanoVax, utilizing the RSV prefusion F protein encapsulated in polyanhydride nanoparticles. Here, we show that the intranasal delivery of RSVNanoVax protected outbred mice from viral replication within the lungs when challenged with RSV out to 1 year post-vaccination. Additionally, RSV-specific antibody responses were generated in both the serum and lung tissue and sustained long-term. These results demonstrate that our vaccine is an encouraging candidate for driving long-term protection in the lungs in a genetically diverse population.

RNA Nanovaccine Protects against White Spot Syndrome Virus in Shrimp.

In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Structural Stability and Antigenicity of Universal Equine H3N8 Hemagglutinin Trimer upon Release from Polyanhydride Nanoparticles and Pentablock Copolymer Hydrogels.

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH33) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH33 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH33 and polymeric nanoadjuvant platforms.

Robust efficacy and long-lasting humoral immunity induced by a respiratory syncytial virus prefusion F-based nanoparticle vaccine in genetically diverse animal models

Abstract Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for 7% of deaths in those less than one year of age. Despite multiple exposures to RSV throughout life, natural infection affords only partial protection and there is currently no licensed vaccine against RSV. We developed a vaccine candidate, RSVNanoVax, composed of polyanhydride nanoparticles encapsulating the RSV prefusion F protein and a CpG1668 adjuvant. To assess the efficacy of RSVNanoVax we utilized two model systems with increased genetic variability, Swiss Webster mice and the cotton rat. Following an intranasal prime-boost vaccination with RSVNanoVax, Swiss Webster mice exhibited robust titers of RSV F-directed IgG antibodies in both the serum and lung tissue that were sustained out to at least 1-year post-vaccination. RSV F-directed IgA was also induced within the lung tissue and nasal passages, suggesting that RSVNanoVax stimulates long-lasting systemic and mucosal antibody responses in outbred mice. Serum antibodies maintained robust neutralizing activity against both RSV A and B virus strains. Following RSV challenge, vaccinated mice exhibited complete viral clearance in the lungs as early as day 2 post-infection. Further studies performed in cotton rats demonstrated that RSVNanoVax conferred protection from both viral replication in the lungs as well as RSV-induced pulmonary immunopathology following viral challenge. Vaccinated cotton rats also exhibited robust titers of RSV F-directed serum antibodies with neutralizing capacity. Overall, RSVNanoVax represents a promising RSV vaccine candidate capable of providing long-term protection and immunity in genetically diverse populations. Supported by grants from NIH (R01 AI124093, T32 CA078586, T32 AI007485)

Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates

Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.

Functionalized polyanhydride nanoparticles for improved treatment of mitochondrial dysfunction.

Parkinson's disease (PD) is a devastating neurodegenerative disease affecting a large proportion of older adults. Exposure to pesticides like rotenone is a leading cause for PD. To reduce disease progression and prolong life expectancy, it is important to target disease mechanisms that contribute to dopaminergic neuronal atrophy, including mitochondrial dysfunction. Achieving targeted mitochondrial delivery is difficult for many therapeutics by themselves, necessitating higher therapeutic doses that could lead to toxicity. To minimize this adverse effect, targeted nano-carriers such as polyanhydride nanoparticles (NPs) can protect therapeutics from degradation and provide sustained release, enabling fewer administrations and lower therapeutic dose. This work expands upon the use of the polyanhydride NP platform for targeted drug delivery by functionalizing the polymer with a derivative of triphenylphosphonium called (3-carboxypropyl) triphenylphosphonium (CPTP) using a novel method that enables longer CPTP persistence on the NPs. The extent to which neurons internalized both nonfunctionalized and functionalized NPs was tested. Next, the efficacy of these nanoformulations in treating rotenone-induced mitochondrial dysfunction in the same cell line was evaluated using a novel neuroprotective drug, mito-metformin. CPTP functionalization significantly improved NP internalization by neuronal cells. This was correlated with significant protection by CPTP-functionalized, mito-metformin encapsulated NPs against rotenone-induced mitochondrial dysfunction. However, nonfunctionalized, mito-metformin encapsulated NPs and soluble mito-metformin administered at the same dose did not significantly protect cells from rotenone-induced toxicity. These results indicate that the targeted NP platform can provide enhanced dose-sparing and potentially reduce the occurrence of systemic side-effects for PD therapeutics.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1α) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1α would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1α systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1α-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1α-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.

Prefusion F-Based Polyanhydride Nanovaccine Induces Both Humoral and Cell-Mediated Immunity Resulting in Long-Lasting Protection against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in both young children and in older adults. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is currently available. We have developed a novel RSV vaccine composed of a prefusion-stabilized variant of the fusion (F) protein (DS-Cav1) and a CpG oligodeoxynucleotide adjuvant encapsulated within polyanhydride nanoparticles, termed RSVNanoVax. A prime-boost intranasal administration of RSVNanoVax in BALB/c mice significantly alleviated weight loss and pulmonary dysfunction in response to an RSV challenge, with protection maintained up to at least 6 mo postvaccination. In addition, vaccinated mice exhibited rapid viral clearance in the lungs as early as 2 d after RSV infection in both inbred and outbred populations. Vaccination induced tissue-resident memory CD4 and CD8 T cells in the lungs, as well as RSV F-directed neutralizing Abs. Based on the robust immune response elicited and the high level of durable protection observed, our prefusion RSV F nanovaccine is a promising new RSV vaccine candidate.

Prefusion F-based polyanhydride nanovaccine induces robust humoral and cell-mediated immunity resulting in long-lasting protection against respiratory syncytial virus

Abstract Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for 7% of deaths in those less than one year of age and leading to more than $300 million in annual medical costs. As natural exposure to the virus generates insufficient immunity, there is a critical need for a safe and efficacious vaccine to prevent RSV-induced disease. We developed an intranasal vaccine, termed RSVNanoVax, composed of polyanhydride nanoparticles encapsulating the RSV prefusion F protein and a CpG1668 oligodeoxynucleotide adjuvant. RSVNanoVax prime-boost vaccination of BALB/c mice induced RSV F-directed IgG serum antibodies specific for both site 0 and site II on the F protein. Systemic antibodies maintained a robust neutralizing capacity out to at least six months post-vaccination. Vaccination with RSVNanoVax also induced F-directed IgA titers within the lungs and nasal passages as well as lung tissue-resident memory CD4 and CD8 T cells, indicating that the vaccine established both a humoral and cell-mediated immune response within the mucosal tissues. Vaccination with RSVNanoVax resulted in protection from RSV-induced disease out to at least six months post-challenge, as measured by weight loss and pulmonary dysfunction. RSVNanoVax vaccination of outbred Swiss Webster mice also elicited protection from RSV-induced disease, and resulted in complete viral clearance as early as day two after RSV challenge. High RSV F-directed antibody titers were also observed in the serum of outbred mice. Based on the robust immune response and the high level of protection observed in both inbred and outbred populations, our prefusion RSV F nanoparticle formulation represents a promising RSV vaccine candidate.

Biodistribution of degradable polyanhydride particles in Aedes aegypti tissues.

Insecticide resistance poses a significant threat to the control of arthropods that transmit disease agents. Nanoparticle carriers offer exciting opportunities to expand the armamentarium of insecticides available for public health and other pests. Most chemical insecticides are delivered by contact or feeding, and from there must penetrate various biological membranes to reach target organs and kill the pest organism. Nanoparticles have been shown to improve bioactive compound navigation of such barriers in vertebrates, but have not been well-explored in arthropods. In this study, we explored the potential of polyanhydride micro- and nanoparticles (250 nm– 3 μm), labeled with rhodamine B to associate with and/or transit across insect biological barriers, including the cuticle, epithelium, midgut and ovaries, in female Ae. aeygpti mosquitoes. Mosquitoes were exposed using conditions to mimic surface contact with a residual spray or paint, topical exposure to mimic contact with aerosolized insecticide, or per os in a sugar meal. In surface contact experiments, microparticles were sometimes observed in association with the exterior of the insect cuticle. Nanoparticles were more uniformly distributed across exterior tissues and present at higher concentrations. Furthermore, by surface contact, topical exposure, or per os, particles were detected in internal organs. In every experiment, amphiphilic polyanhydride nanoparticles associated with internal tissues to a higher degree than hydrophobic nanoparticles. In vitro, nanoparticles associated with Aedes aegypti Aag2 cells within two hours of exposure, and particles were evident in the cytoplasm. Further studies demonstrated that particle uptake is dependent on caveolae-mediated endocytosis. The propensity of these nanoparticles to cross biological barriers including the cuticle, to localize in target tissue sites of interest, and to reach the cytoplasm of cells, provides great promise for targeted delivery of insecticidal candidates that cannot otherwise reach these cellular and subcellular locations.

Polyanhydride Nanoparticles Induce Low Inflammatory Dendritic Cell Activation Resulting in CD8+ T Cell Memory and Delayed Tumor Progression.

IntroductionAdjuvants and immunotherapies designed to activate adaptive immunity to eliminate infectious disease and tumors have become an area of interest aimed at providing a safe and effective strategy to prevent or eliminate disease. Existing approaches would benefit from the development of immunization regimens capable of inducing efficacious cell-mediated immunity directed toward CD8+ T cell-specific antigens. This goal is critically dependent upon appropriate activation of antigen-presenting cells (APCs) most notably dendritic cells (DCs). In this regard, polyanhydride particles have been shown to be effectively internalized by APCs and induce activation.MethodsHere, a prophylactic vaccine regimen designed as a single-dose polyanhydride nanovaccine encapsulating antigen is evaluated for the induction of CD8+ T cell memory in a model system where antigen-specific protection is restricted to CD8+ T cells. Bone marrow-derived dendritic cells (BMDCs) are used as an in vitro model system to evaluate the magnitude and phenotype of APC activation. Primary DCs, particularly those with described ability to activate CD8+ T cells, are also evaluated for their in vitro responses to polyanhydride nanoparticles.ResultsHerein, polyanhydride nanoparticles are shown to induce potent in vitro upregulation of costimulatory molecules on the cell surface of BMDCs. In contrast to the classically used TLR agonists, nanoparticles did not induce large amounts of pro-inflammatory cytokines, did not induce characteristic metabolic response of DCs, nor produce innate antimicrobial effector molecules, such as nitric oxide (NO). The polyanhydride nanovaccine results in protective CD8+ T cell responses as measured by inhibition of tumor progression and survival.DiscussionTogether, these results suggest that the use of a polyanhydride-based nanovaccine can be an effective approach to inducing antigen-specific CD8+ T cell memory by providing antigen delivery and DC activation while avoiding overt inflammatory responses typically associated with traditional adjuvants.

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4β.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

High-Throughput Synthesis and Screening of Rapidly Degrading Polyanhydride Nanoparticles.

Combinatorial techniques can accelerate the discovery and development of polymeric nanodelivery devices by pairing high-throughput synthesis with rapid materials characterization. Biodegradable polyanhydrides demonstrate tunable release, high cellular internalization, and dose sparing properties when used as nanodelivery devices. This nanoparticle platform shows promising potential for small molecule drug delivery, but the pace of understanding and rational design of these nanomedicines is limited by the low throughput of conventional characterization. This study reports the use of a high-throughput method to synthesize libraries of a newly synthesized, rapidly eroding polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and sebacic acid (SA) monomers. The high-throughput method enabled efficient screening of copolymer microstructure, revealing weak block-type and alternating architectures. The high-throughput method was adapted to synthesize nanoparticle libraries encapsulating hydrophobic model drugs. Drug release from these nanoparticles was rapid, with a majority of the payload released within 3 days. Drug release was dramatically slowed at acidic pH, which could be useful for oral drug delivery. Rhodamine B (RhoB) release kinetics generally followed patterns of polymer erosion kinetics, while Coomassie brilliant blue (CBB) released the fastest from the slowest degrading polymer chemistry and vice versa. These differences in trends between copolymer chemistry and release kinetics were hypothesized to arise from differences in mixing thermodynamics. A high-throughput method was developed to synthesize polymer-drug film libraries and characterize mixing thermodynamics by melting point depression. Rhodamine B had a negative χ for all copolymers with <30 mol % CPTEG tested, indicating a tendency toward miscibility. By contrast, CBB χ increased, eventually becoming positive near 15:85 CPTEG:SA, with increasing CPTEG content. This indicates an increasing tendency toward phase separation in CPTEG-rich copolymers. These in vitro results screening polymer-drug interactions showed good agreement with in silico predictions from Hansen solubility parameter estimation and were able to explain the observed differences in model drug release trends.