The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model.

Background: Innovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells. Methods: The immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions. Results: Neutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions. Conclusion: The MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.

Bovine milk-derived cells express transcriptome markers of pluripotency and secrete bioactive factors with regenerative and antimicrobial activity.

The bovine mammary stem/progenitor cell secretome stimulates regeneration in vitro and contains proteins associated with antimicrobial defense. This has led to the exploration of the secretome as a biologic treatment for mastitis, a costly inflammation of the udder commonly caused by bacteria. This study reports on a population of bovine mammary stem/progenitor cells isolated non-invasively from milk (MiDCs). MiDCs were characterized by immunophenotyping, mammosphere formation assays, and single cell RNA sequencing. They displayed epithelial morphology, exhibited markers of mammary stem/progenitor cells, and formed mammospheres, like mammary gland tissue-isolated stem/progenitor cells. Single cell RNA sequencing revealed two sub-populations of MiDCs: epithelial cells and macrophages. Functionally, the MiDC secretome increased fibroblast migration, promoted angiogenesis of endothelial cells, and inhibited the growth of mastitis-associated bacteria, including antibiotic-resistant strains, in vitro. These qualities of MiDCs render them a source of stem cells and stem cell products that may be used to treat diseases affecting the dairy industry, including mastitis.

Epitope-coated polymer particles elicit neutralising antibodies against Plasmodium falciparum sporozoites.

The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.

An inactivated influenza D virus vaccine partially protects cattle from respiratory disease caused by homologous challenge.

Originally isolated from swine, the proposed influenza D virus has since been shown to be common in cattle. Inoculation of IDV to naïve calves resulted in mild respiratory disease histologically characterized by tracheitis. As several studies have associated the presence of IDV with acute bovine respiratory disease (BRD), we sought to investigate the efficacy of an inactivated IDV vaccine. Vaccinated calves seroconverted with hemagglutination inhibition titers 137-169 following two doses. Non-vaccinated calves challenged with a homologous virus exhibited signs of mild respiratory disease from days four to ten post challenge which was significantly different than negative controls at days five and nine post challenge. Peak viral shedding of approximately 5 TCID50/mL was measured in nasal and tracheal swabs and bronchoalveolar lavage fluids four to six days post challenge. Viral titers were significantly (P<0.05) decreased 1.4 TCID50/mL, 3.6 TCID50/mL and 5.0 TCID50/mL, respectively, in the aforementioned samples collected from vaccinated animals compared to non-vaccinated controls at peak shedding. Viral antigen was detected in the respiratory epithelium of the nasal turbinates and trachea by immunohistochemistry from all unvaccinated calves but in significantly fewer vaccinates. Inflammation characterized by neutrophils was observed in the nasal turbinate and trachea but not appreciably in lungs. Together these results support an etiologic role for IDV in BRD and demonstrate that partial protection is afforded by an inactivated vaccine.

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

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

Pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles.

PURPOSE: For the rational design of nanovaccines against respiratory pathogens, careful selection of optimal particle size and chemistry is paramount. This work investigates the impact of these properties on the deposition, biodistribution, and cellular interactions of nanoparticles within the lungs. METHOD: In this work, biodegradable poly(sebacic anhydride) (poly(SA)) nanoparticles of multiple sizes were synthesized with narrow particle size distributions. The lung deposition and retention as well as the internalization by phagocytic cells of these particles were compared to that of non-degradable monodisperse polystyrene nanoparticles of similar sizes. RESULTS: The initial deposition of intranasally administered particles in the lungs was dependent on primary particle size, with maximal deposition occurring for the 360-470 nm particles, regardless of chemistry. Over time, both particle size and chemistry affected the frequency of particle-positive cells and the specific cell types taking up particles. The biodegradable poly(SA) particles associated more closely with phagocytic cells and the dynamics of this association impacted the clearance of these particles from the lung. CONCLUSIONS: The findings reported herein indicate that both size and chemistry control the fate of intranasally administered particles and that the dynamics of particle association with phagocytic cells in the lungs provide important insights for the rational design of pulmonary vaccine delivery vehicles.

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

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

Vaccine technologies against avian influenza: current approaches and new directions.

The potential epidemiological human pandemic resulting from highly pathogenic avian influenza (HPAI) H5N1 has been studied extensively since the identification of the virus in the Guangdong province of China. The majority of research has focused on the unique and severe histopathological lesions induced by the virus. The severe pathological presentation of these infections has also prompted interest in identifying preventive and therapeutic approaches against HPAI. The potential severity of a HPAI pandemic and the efforts to identify effective intervention strategies have led to many novel discoveries in vaccine and antiviral development that are critically examined in this review.

Combinatorial evaluation of in vivo distribution of polyanhydride particle-based platforms for vaccine delivery.

Several challenges are associated with current vaccine strategies, including repeated immunizations, poor patient compliance, and limited approved routes for delivery, which may hinder induction of protective immunity. Thus, there is a need for new vaccine adjuvants capable of multi-route administration and prolonged antigen release at the site of administration by providing a depot within tissue. In this work, we designed a combinatorial platform to investigate the in vivo distribution, depot effect, and localized persistence of polyanhydride nanoparticles as a function of nanoparticle chemistry and administration route. Our observations indicated that the route of administration differentially affected tissue residence times. All nanoparticles rapidly dispersed when delivered intranasally but provided a depot when administered parenterally. When amphiphilic and hydrophobic nanoparticles were administered intranasally, they persisted within lung tissue. These results provide insights into the chemistry- and route-dependent distribution and tissue-specific association of polyanhydride nanoparticle-based vaccine adjuvants.

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

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

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

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

Harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform. In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AMɸ) predominate in the lung parenchyma. AMɸ are highly efficient in clearing the lungs of microbial pathogens and cell debris. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response. AMɸ also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime naïve T cells. A relatively pure population of AMɸ (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AMɸ harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AMɸ from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.