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.

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.

Effective antiviral coatings for deactivating SARS-CoV-2 virus on N95 respirator masks or filters.

The application of antiviral coatings to masks and respirators is a potential mitigating step toward reducing viral transmission during the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The use of appropriate masks, social distancing, and vaccines is the immediate solution for limiting the viral spread and protecting people from this virus. N95 respirator masks are effective in filtering the virus particles, but they cannot kill or deactivate the virus. We report a possible approach to deactivating SARS-CoV-2 by applying an antimicrobial coating (Goldshield 75) to masks and respirators, rendering them suitable for repeated use. Masks coated with Goldshield 75 demonstrated continuous inactivation of the Alpha and Beta variants of the SARS-CoV-2 over a 3-day period and no loss of inactivation when stored at temperatures at 50 °C.

Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery.

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells' luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2-3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.

Single-dose combination nanovaccine induces both rapid and long-lived protection against pneumonic plague.

Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this pathogen; however, USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to induce protection against pneumonic plague. Many F1-V-based vaccine formulations require prime-boost immunization to achieve protective immunity, and there are limited reports of rapid induction of protective immunity (≤ 14 days post-immunization (DPI)). The STimulator of INterferon Genes agonists cyclic dinucleotides (CDNs) have been shown to be promising vaccine adjuvants. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also shown to enhance immune responses due to their dual functionality as adjuvants and delivery vehicles. In this work, a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined with the CDN, dithio-RP,RP-cyclic di-guanosine monophosphate, to induce rapid and long-lived protective immunity against pneumonic plague. All mice immunized with a single dose combination nanovaccine were protected from Y. pestis lethal challenge within 14 DPI and demonstrated enhanced protection over F1-V adjuvanted with CDNs alone at challenge doses ≥7000 CFU Y. pestis CO92. In addition, 75% of mice receiving the single dose of the combination nanovaccine were protected from challenge at 182 DPI, while maintaining high levels of antigen-specific serum IgG. ELISPOT analysis of vaccinated animals at 218 DPI revealed F1-V-specific long-lived plasma cells in bone marrow in mice vaccinated with CDN adjuvanted F1-V or the combination nanovaccine. Microarray analysis of serum from these vaccinated mice revealed the presence of serum antibody that bound to a broad range of F1 and V linear epitopes. These results demonstrate that combining the adjuvanticity of CDNs with a nanovaccine delivery system enables induction of both rapid and long-lived protective immunity against Y. pestis. STATEMENT OF SIGNIFICANCE: • Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this biodefense pathogen. • We designed a combination nanovaccine comprising of F1-V antigen-loaded polyanhydride nanoparticles and a cyclic dinucleotide adjuvant to induce both rapid and long-lived protective immunity against pneumonic plague. • Animals immunized with the combination nanovaccine maintained high levels of antigen-specific serum IgG and long-lived plasma cells in bone marrow and the serum antibody showed a high affinity for a broad range of F1 and V linear epitopes. • The combination nanovaccine is a promising next-generation vaccine platform against weaponized Y. pestis based on its ability to induce both rapid and long-lived protective immunity.

Vitamin C and B3 as new biomaterials to alter intestinal stem cells.

Vitamin C (ascorbic acid) and vitamin B3 (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact toward homeostasis and epigenetic changes (D'Aniello et al., Stem Cells International, 2017, 1-16). Vitamin B3 aids in maintaining healthy intestinal homeostasis and reducing gut inflammation by participating in the rapamycin signaling pathway (Kumar et al., The American Journal of Physiology-Gastrointestinal and Liver Physiology, 2013). In this study, vitamin C and vitamin B3 (600 and 1,200 µg/mL) have been explored as potential new biomaterials to study their effects on four types of intestinal stem cells which are isolated from mice bearing different microbiota. We observed that C3H ASF and 129 ASF IL-10 are more sensitive towardB7 600 µg/mL vitamin B3 and 1,200 µg/mL vitamin C. The lowest growth rate and viability for all types of organoids was with 1,200 µg/mL vitamin C. From quantitative polymerase chain reaction analysis (qPCR analysis), MUC2 was upregulated for 129 ASF and C3H Conv when exposed to 600 µg/mL and 1,200 µg/mL vitamin C. It suggests that large amounts of glycoprotein may be produced after adding high concentrations of vitamin C. Since inflammatory bowel disease has low level of MUC2, this finding may be helpful in restoring mucosal health by upregulating the MUC2 gene while altering patient's microbiota (Sibila et al., Annals of the American Thoracic Society, 2016). These results are expected to have a positive translational impact because this bottom-up strategy would be instrumental in developing Vitamin C and B3 based orally available therapeutic strategies and formula for advancing the fields of gastrointestinal regenerative medicine.

Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release.

Drug delivery vehicles can improve the functional efficacy of existing antimicrobial therapies by improving biodistribution and targeting. A critical property of such nanomedicine formulations is their ability to control the release kinetics of their payloads. The combination of (and interactions among) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward the rational design of nanomedicines by identifying key descriptors and correlating them to nanoparticle release behavior. In this work, antibiotic release kinetics data were generated from polyanhydride nanoparticle formulations with varying copolymer compositions, encapsulated drug type, and drug loading. Four antibiotics, doxycycline, rifampicin, chloramphenicol, and pyrazinamide, were used. Linear manifold learning methods were used to relate drug release properties with polymer, drug, and nanoparticle properties, and key descriptors were identified that are highly correlated with release properties. However, these linear methods could not predict release behavior. Nonlinear multivariate modeling based on graph theory was then used to deconvolute the governing relationships between these properties, and predictive models were generated to rapidly screen lead nanomedicine formulations with desirable release properties with minimal nanoparticle characterization. Release kinetics predictions of two drugs containing atoms not included in the model showed good agreement with experimental results, validating the model and indicating its potential to virtually explore new polymer and drug pairs not included in the training data set. The models were shown to be robust after the inclusion of these new formulations, in that the new inclusions did not significantly change model regression. This approach provides the first step toward the development of a framework that can be used to rationally design nanomedicine formulations by selecting the appropriate carrier for a drug payload to program desirable release kinetics.

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri.

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75-3.0 µg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri-infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.

Nanotherapeutic provides dose sparing and improved antimicrobial activity against Brucella melitensis infections.

New therapies are needed to treat chronic bacterial diseases and intracellular pathogens, in particular, are very difficult to manage. The use of nanotherapeutics represents an approach to exploit size and charge of biological membranes to overcome barriers for treatment of intracellular pathogens including Brucella melitensis. In this work, polyanhydride nanoparticles comprised of copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were synthesized to encapsulate antimicrobial compounds doxycycline and rifampicin. The nanoparticles demonstrated sustained release of rifampicin for a week with the antimicrobial activity peaking at 72 h and lasting up to a week. Treatment of B. melitensis infected macrophages with rifampicin-containing nanoparticles rapidly eliminated viable intracellular bacteria following 48 h of treatment and pretreatment with the nano-formulations prevented intracellular infection in contrast to soluble drug controls. Treatment of infected BALB/c mice with a nanoparticle cocktail containing doxycycline and rifampicin for five days decreased bacterial burden by three log10 in the liver. Extended release of antibiotics was demonstrated in vivo by treating B. melitensis infected mice with the standard therapy of daily 0.5 mg doxycycline dose or single 0.5 mg doxycycline-encapsulated nanoparticles delivered once a week. After 3 weeks, bacterial counts in spleen and liver were statistically equal between animals treated with the weekly nano-formulation and daily soluble drug, representing a seven-fold dose sparing. Altogether, these results demonstrated that the use of nanotherapeutics was successful at increasing antimicrobial efficacy and improving in vivo activity through a combination of intracellular delivery, dose sparing, and extended release in treating chronic bacterial infections. This platform technology can also provide benefits for drug delivery against other chronic intracellular bacterial pathogens, including Mycobacterium and Burkholderia species, including treatments against antibiotic-resistant infections.

Ex Vivo Study of Telluride Nanowires in Minigut.

Compound semiconductor nanomaterials, such as telluride nanowires, nanorods, and nanoparticles, have many unique properties for wide range of potential applications. The interaction between organoids (a biological system) and telluride nanowires is a crucial research area for human health in terms of its safety concerns. In this study, we demonstrated a case study on Bi2Te3 nanowires. Through live/dead cell viability testing, bright-light image analysis, and surface area calculations, we showed that 50 µg/mL Bi2Te3 exerts minimum influence on shrinking crypts and disrupting lumen structure, which causes unhealthy growth. Within this optimal concentration, Bi2Te3 nanowires can stay as a stable and non-toxic material inside the intestine. Unlike the previous studies of the cytotoxicity of Telluride nanomaterials interacting with single type of cells, our research demonstrated the first study of the interactions of engineered Telluride nanomaterials with a real complex gastrointestinal tract system as our primary small intestinal crypts were directly isolated from mice and grew into a self-renewable system with various types of cells and different cell pathways, which has the capability to mimic a fully functional intestinal epithelium layer for a realistic study inside the gastrointestinal tract. Most importantly, we showed that Bi2Te3 nanowires, under infrared exposure, can act as a potential shield to stimulate cell viability and improve cell survivability.

Intestinal organoids containing poly(lactic-co-glycolic acid) nanoparticles for the treatment of inflammatory bowel diseases.

Inflammatory bowel disease (IBD) causes inflammation to the gastrointestinal tract. Local administration of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) can alleviate the symptoms of IBD. The application of nanoparticles for IBD treatment in direct rectal administration showed high drug availability and treatment efficacy. However, relying on size-dependent adsorption of smaller particles is not sufficient for making the formulation capable of targeting. Intestinal organoids can improve the functionality of the nanoparticles due to their ability to adsorb small nanoparticle inside the lumen and attach to the damaged area. In this study, intestinal organoids were used as carriers of 5-ASA-loaded poly(lactic-co-glycolic acid) nanoparticles. The nanoparticle sizes, confirmed by scanning electron microscopy, were 200-300 nm and the zeta potential were negative. The nanoparticles did not have any noticeable pernicious effect on organoid growth and viability. After mixing the nanoparticles with Matrigel and organoids, Rhodamine B loaded inside the nanoparticles was highly detected inside the organoid's lumen after 3 days by confocal fluorescent microscopy and no longer detected in the lumen after day 4. It may be attributed to the ability of the lumen to digest particles. Thus, the organoid Trojan horse system is a possible approach for delivering drugs to inflamed areas. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 876-886, 2018.