Next-generation nanovaccine induces durable immunity and protects against SARS-CoV-2.

While first generation SARS-CoV-2 vaccines were effective in slowing the spread and severity of disease during the COVID-19 pandemic, there is a need for vaccines capable of inducing durable and broad immunity against emerging variants of concern. Nanoparticle-based vaccines (i.e., "nanovaccines") composed of polyanhydride nanoparticles and pentablock copolymer micelles have previously been shown to protect against respiratory pathogens, including influenza A virus, respiratory syncytial virus, and Yersinia pestis. In this work, a nanovaccine containing SARS-CoV-2 spike and nucleocapsid antigens was designed and optimized. The optimized nanovaccine induced long-lived systemic IgG antibody responses against wild-type SARS-CoV-2 virus. In addition, the nanovaccine induced antibody responses capable of neutralization and cross-reactivity to multiple SARS-CoV-2 variants (including B.1.1.529) and antigen-specific CD4+ and CD8+ T cell responses. Finally, the nanovaccine protected mice against a lethal SARS-CoV-2 challenge, setting the stage for advancing particle-based SARS-CoV-2 nanovaccines. STATEMENT OF SIGNIFICANCE: First-generation SARS-CoV-2 vaccines were effective in slowing the spread and limiting the severity of COVID-19. However, current vaccines target only one antigen of the virus (i.e., spike protein) and focus on the generation of neutralizing antibodies, which may be less effective against new, circulating strains. In this work, we demonstrated the ability of a novel nanovaccine platform, based on polyanhydride nanoparticles and pentablock copolymer micelles, to generate durable and broad immunity against SARS-CoV-2. These nanovaccines induced long-lasting (> 62 weeks) serum antibody responses which neutralized binding to ACE2 receptors and were cross-reactive to multiple SARS-CoV-2 variants. Additionally, mice immunized with the SARS-CoV-2 nanovaccine showed a significant increase of antigen-specific T cell responses in the draining lymph nodes and spleens. Together, these nanovaccine-induced immune responses contributed to the protection of mice against a lethal challenge of live SARS-CoV-2 virus, indicating that this nanovaccine platform is a promising next-generation SARS-CoV-2 vaccine.

Polyanhydride nanovaccine against H3N2 influenza A virus generates mucosal resident and systemic immunity promoting protection.

Influenza A virus (IAV) causes significant morbidity and mortality worldwide due to seasonal epidemics and periodic pandemics. The antigenic drift/shift of IAV continually gives rise to new strains and subtypes, aiding IAV in circumventing previously established immunity. As a result, there has been substantial interest in developing a broadly protective IAV vaccine that induces, durable immunity against multiple IAVs. Previously, a polyanhydride nanoparticle-based vaccine or nanovaccine (IAV-nanovax) encapsulating H1N1 IAV antigens was reported, which induced pulmonary B and T cell immunity and resulted in cross-strain protection against IAV. A key feature of IAV-nanovax is its ability to easily incorporate diverse proteins/payloads, potentially increasing its ability to provide broad protection against IAV and/or other pathogens. Due to human susceptibility to both H1N1 and H3N2 IAV, several H3N2 nanovaccines were formulated herein with multiple IAV antigens to examine the "plug-and-play" nature of the polyanhydride nanovaccine platform and determine their ability to induce humoral and cellular immunity and broad-based protection similar to IAV-nanovax. The H3N2-based IAV nanovaccine formulations induced systemic and mucosal B cell responses which were associated with antigen-specific antibodies. Additionally, systemic and lung-tissue resident CD4 and CD8 T cell responses were enhanced post-vaccination. These immune responses corresponded with protection against both homologous and heterosubtypic IAV infection. Overall, these results demonstrate the plug-and-play nature of the polyanhydride nanovaccine platform and its ability to generate immunity and protection against IAV utilizing diverse antigenic payloads.

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.

Synthesis and Optimization of Next-Generation Low-Molecular-Weight Pentablock Copolymer Nanoadjuvants.

Polymeric nanomaterials such as Pluronic®-based pentablock copolymers offer important advantages over traditional vaccine adjuvants and have been increasingly investigated in an effort to develop more efficacious vaccines. Previous work with Pluronic® F127-based pentablock copolymers, functionalized with poly(diethyl aminoethyl methacrylate) (PDEAEM) blocks, demonstrated adjuvant capabilities through the antigen presentation and crosslinking of B cell receptors. In this work, we describe the synthesis and optimization of a new family of low-molecular-weight Pluronic®-based pentablock copolymer nanoadjuvants with high biocompatibility and improved adjuvanticity at low doses. We synthesized low-molecular-weight Pluronic® P123-based pentablock copolymers with PDEAEM blocks and investigated the relationship between polymer concentration, micellar size, and zeta potential, and measured the release kinetics of a model antigen, ovalbumin, from these nanomaterials. The Pluronic® P123-based pentablock copolymer nanoadjuvants showed higher biocompatibility than the first-generation Pluronic® F127-based pentablock copolymer nanoadjuvants. We assessed the adjuvant capabilities of the ovalbumin-containing Pluronic® P123-based pentablock copolymer-based nanovaccines in mice, and showed that animals immunized with these nanovaccines elicited high antibody titers, even when used at significantly reduced doses compared to Pluronic® F127-based pentablock copolymers. Collectively, these studies demonstrate the synthesis, self-assembly, biocompatibility, and adjuvant properties of a new family of low-molecular-weight Pluronic® P123-based pentablock copolymer nanomaterials, with the added benefits of more efficient renal clearance, high biocompatibility, and enhanced adjuvanticity at low polymer concentrations.

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.

Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection.

Human respiratory syncytial virus (HRSV) is a leading cause of death in young children and there are no FDA approved vaccines. Bovine RSV (BRSV) is antigenically similar to HRSV, and the neonatal calf model is useful for evaluation of HRSV vaccines. Here, we determined the efficacy of a polyanhydride-based nanovaccine encapsulating the BRSV post-fusion F and G glycoproteins and CpG, delivered prime-boost via heterologous (intranasal/subcutaneous) or homologous (intranasal/intranasal) immunization in the calf model. We compared the performance of the nanovaccine regimens to a modified-live BRSV vaccine, and to non-vaccinated calves. Calves receiving nanovaccine via either prime-boost regimen exhibited clinical and virological protection compared to non-vaccinated calves. The heterologous nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA, and induced similar clinical, virological and pathological protection as the commercial modified-live vaccine. Principal component analysis identified BRSV-specific humoral and cellular responses as important correlates of protection. The BRSV-F/G CpG nanovaccine is a promising candidate vaccine to reduce RSV disease burden in humans and animals.

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

BACKGROUND: Age-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. RESULTS: In 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. CONCLUSIONS: Overall, 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.

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: These 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.

Data analytics-guided rational design of antimicrobial nanomedicines against opportunistic, resistant pathogens.

Nanoparticle carriers can improve antibiotic efficacy by altering drug biodistribution. However, traditional screening is impracticable due to a massive dataspace. A hybrid informatics approach was developed to identify polymer, antibiotic, and particle determinants of antimicrobial nanomedicine activity against Burkholderia cepacia, and to model nanomedicine performance. Polymer glass transition temperature, drug octanol-water partition coefficient, strongest acid dissociation constant, physiological charge, particle diameter, count and mass mean polydispersity index, zeta potential, fraction drug released at 2 h, and fraction release slope at 2 h were highly correlated with antimicrobial performance. Graph analysis provided dimensionality reduction while preserving nonlinear descriptor-property relationships, enabling accurate modeling of nanomedicine performance. The model successfully predicted particle performance in holdout validation, with moderate accuracy at rank-ordering. This data analytics-guided approach provides an important step toward the development of a rational design framework for antimicrobial nanomedicines against resistant infections by selecting appropriate carriers and payloads for improved potency.

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.

Delivering an Immunocastration Vaccine via a Novel Subcutaneous Implant.

Immunocastration relies on the vaccine-mediated stimulation of an immune response to gonadotropin-releasing hormone (GnRH) in order to interrupt spermatogenesis. This approach offers a less painful alternative to traditional castration approaches but the current, commercially available options require multiple doses of vaccine to maintain sterility. Thus, a series of pilot studies were conducted to determine the feasibility of a single-dose immunocastration vaccine implant. These five studies utilized a total of 44 Holstein bulls to determine the optimal vaccine composition and validate the ability of a stainless-steel subcutaneous implant to deliver a vaccine. Outcome measures included the duration of implant retention, scrotal dimensions and temperature, implant site temperature, anti-GnRH antibodies, and serum testosterone concentration. Over the course of several studies, anti-GnRH antibodies were successfully stimulated by vaccine implants. No significant treatment effects on scrotal dimensions or testosterone were detected over time, but changes in spermatogenesis were detected across treatment groups. Results indicate that a single-dose implantable immunocastration vaccine elicits a humoral immune response and could impact spermatogenesis in bulls. These findings provide opportunities for the refinement of this technology to improve implant retention over longer periods of time. Taken together, this approach will offer producers and veterinarians an alternative to physical castration methods, to improve animal welfare during routine livestock management procedures.

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.

Nanocarriers for pancreatic cancer imaging, treatments, and immunotherapies.

Pancreatic tumors are highly desmoplastic and immunosuppressive. Delivery and distribution of drugs within pancreatic tumors are compromised due to intrinsic physical and biochemical stresses that lead to increased interstitial fluid pressure, vascular compression, and hypoxia. Immunotherapy-based approaches, including therapeutic vaccines, immune checkpoint inhibition, CAR-T cell therapy, and adoptive T cell therapies, are challenged by an immunosuppressive tumor microenvironment. Together, extensive fibrosis and immunosuppression present major challenges to developing treatments for pancreatic cancer. In this context, nanoparticles have been extensively studied as delivery platforms and adjuvants for cancer and other disease therapies. Recent advances in nanotechnology have led to the development of multiple nanocarrier-based formulations that not only improve drug delivery but also enhance immunotherapy-based approaches for pancreatic cancer. This review discusses and critically analyzes the novel nanoscale strategies that have been used for drug delivery and immunomodulation to improve treatment efficacy, including newly emerging immunotherapy-based approaches. This review also presents important perspectives on future research directions that will guide the rational design of novel and robust nanoscale platforms to treat pancreatic tumors, particularly with respect to targeted therapies and immunotherapies. These insights will inform the next generation of clinical treatments to help patients manage this debilitating disease and enhance survival rates.

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.

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.

Biomaterial nanocarrier-driven mechanisms to modulate anti-tumor immunity.

Cancer immunotherapy approaches that utilize or enhance patients' inherent immunity have received extensive attention in the past decade. Biomaterial-based nanocarriers with tunable physicochemical properties offer significant promise in cancer immunotherapies. They can lower payload toxicity, provide sustained release of diverse payloads, and target specific disease site(s). Furthermore, nanocarrier-mediated immunotherapies can induce antigen-specific T lymphocytes, tissue-directed immune activation, and apoptosis of cancer cells all of which may comprise a new paradigm in cancer immunotherapy. This review describes key steps in biomaterial-mediated immune activation ranging from biomaterial surface protein adsorption, antigen presenting cell processing, and T cell activation. Nanocarrier-based immunomodulatory mechanisms including inherent adjuvanticity, enhanced cellular internalization, lymph node delivery, cross-presentation, and immunogenic cell death are discussed. In addition, studies that synergistically influence outcomes of nanocarrier-based combination immunotherapies are presented.

Self-assembling synthetic nanoadjuvant scaffolds cross-link B cell receptors and represent new platform technology for therapeutic antibody production.

Host antibody responses are pivotal for providing protection against infectious agents. We have pioneered a new class of self-assembling micelles based on pentablock copolymers that enhance antibody responses while providing a low inflammatory environment compared to traditional adjuvants. This type of "just-right" immune response is critical in the rational design of vaccines for older adults. Here, we report on the mechanism of enhancement of antibody responses by pentablock copolymer micelles, which act as scaffolds for antigen presentation to B cells and cross-link B cell receptors, unlike other micelle-forming synthetic block copolymers. We exploited this unique mechanism and developed these scaffolds as a platform technology to produce antibodies in vitro. We show that this novel approach can be used to generate laboratory-scale quantities of therapeutic antibodies against multiple antigens, including those associated with SARS-CoV-2 and Yersinia pestis, further expanding the value of these nanomaterials to rapidly develop countermeasures against infectious diseases.

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.