Inhibiting the cGAS-STING Pathway in Ulcerative Colitis with Programmable Micelles.

Ulcerative colitis is a chronic condition in which a dysregulated immune response contributes to the acute intestinal inflammation of the colon. Current clinical therapies often exhibit limited efficacy and undesirable side effects. Here, programmable nanomicelles were designed for colitis treatment and loaded with RU.521, an inhibitor of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. STING-inhibiting micelles (SIMs) comprise hyaluronic acid-stearic acid conjugates and include a reactive oxygen species (ROS)-responsive thioketal linker. SIMs were designed to selectively accumulate at the site of inflammation and trigger drug release in the presence of ROS. Our in vitro studies in macrophages and in vivo studies in a murine model of colitis demonstrated that SIMs leverage HA-CD44 binding to target sites of inflammation. Oral delivery of SIMs to mice in both preventive and delayed therapeutic models ameliorated colitis's severity by reducing STING expression, suppressing the secretion of proinflammatory cytokines, enabling bodyweight recovery, protecting mice from colon shortening, and restoring colonic epithelium. In vivo end points combined with metabolomics identified key metabolites with a therapeutic role in reducing intestinal and mucosal inflammation. Our findings highlight the significance of programmable delivery platforms that downregulate inflammatory pathways at the intestinal mucosa for managing inflammatory bowel diseases.

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.

The E. coli pathobiont LF82 encodes a unique variant of σ70 that results in specific gene expression changes and altered phenotypes.

LF82, an adherent invasive Escherichia coli pathobiont, is associated with ileal Crohn's disease, an inflammatory bowel disease of unknown etiology. Although LF82 contains no virulence genes, it carries several genetic differences, including single nucleotide polymorphisms (SNPs), that distinguish it from nonpathogenic E. coli. We have identified and investigated an extremely rare SNP that is within the highly conserved rpoD gene, encoding σ70, the primary sigma factor for RNA polymerase. We demonstrate that this single residue change (D445V) results in specific transcriptome and phenotypic changes that are consistent with multiple phenotypes observed in LF82, including increased antibiotic resistance and biofilm formation, modulation of motility, and increased capacity for methionine biosynthesis. Our work demonstrates that a single residue change within the bacterial primary sigma factor can lead to multiple alterations in gene expression and phenotypic changes, suggesting an underrecognized mechanism by which pathobionts and other strain variants with new phenotypes can emerge.

Pathway Tools Management of Pathway/Genome Data for Microbial Communities.

The Pathway Tools (PTools) software provides a suite of capabilities for storing and analyzing integrated collections of genomic and metabolic information in the form of organism-specific Pathway/Genome Databases (PGDBs). A microbial community is represented in PTools by generating a PGDB from each metagenome-assembled genome (MAG). PTools computes a metabolic reconstruction for each organism, and predicts its operons. The properties of individual MAGs can be investigated using the many search and visualization operations within PTools. PTools also enables the user to investigate the properties of the microbial community by issuing searches across the full community, and by performing comparative operations across genome and pathway information. The software can generate a metabolic network diagram for the community, and it can overlay community omics datasets on that network diagram. PTools also provides a tool for searching for metabolic transformation routes across an organism community.

Resources to Facilitate Use of the Altered Schaedler Flora (ASF) Mouse Model to Study Microbiome Function.

Animals colonized with a defined microbiota represent useful experimental systems to investigate microbiome function. The altered Schaedler flora (ASF) represents a consortium of eight murine bacterial species that have been used for more than 4 decades where the study of mice with a reduced microbiota is desired. In contrast to germ-free mice, or mice colonized with only one or two species, ASF mice show the normal gut structure and immune system development. To further expand the utility of the ASF, we have developed technical and bioinformatic resources to enable a systems-based analysis of microbiome function using this model. Here, we highlighted four distinct applications of these resources that enable and improve (i) measurements of the abundance of each ASF member by quantitative PCR; (ii) exploration and comparative analysis of ASF genomes and the metabolic pathways they encode that comprise the entire gut microbiome; (iii) global transcriptional profiling to identify genes whose expression responds to environmental changes within the gut; and (iv) discovery of genetic changes resulting from the evolutionary adaptation of the microbiota. These resources were designed to be accessible to a broad community of researchers that, in combination with conventionally-reared mice (i.e., with complex microbiome), should contribute to our understanding of microbiome structure and function. IMPORTANCE Improved experimental systems are needed to advance our understanding of how the gut microbiome influences processes of the mammalian host as well as microbial community structure and function. An approach that is receiving considerable attention is the use of animal models that harbor a stable microbiota of known composition, i.e., defined microbiota, which enables control over an otherwise highly complex and variable feature of mammalian biology. The altered Schaedler flora (ASF) consortium is a well-established defined microbiota model, where mice are stably colonized with 8 distinct murine bacterial species. To take better advantage of the ASF, we established new experimental and bioinformatics resources for researchers to make better use of this model as an experimental system to study microbiome function.

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.

Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in life.

The gastrointestinal microbiota begins to be acquired at birth and continually matures through early adolescence. Despite the relevance for gut health, few studies have evaluated the impact of pathobiont colonization of neonates on the severity of colitis later in life. LF82 is an adherent invasive E. coli strain associated with ileal Crohn's disease. The aim of this study was to evaluate the severity of dextran sodium sulfate (DSS)-induced colitis in mice following E. coli LF82 colonization. Gnotobiotic mice harboring the altered Schaedler flora (ASF) were used as the model. While E. coli LF82 is neither adherent nor invasive, it was been demonstrated that adult ASF mice colonized with E. coli LF82 develop more severe DSS-induced colitis compared to control ASF mice treated with DSS. Therefore, we hypothesized that E. coli LF82 colonization of neonatal ASF mice would reduce the severity of DSS-induced inflammation compared to adult ASF mice colonized with E. coli LF82. To test this hypothesis, adult ASF mice were colonized with E. coli LF82 and bred to produce offspring (LF82N) that were vertically colonized with LF82. LF82N and adult-colonized (LF82A) mice were given 2.0% DSS in drinking water for seven days to trigger colitis. More severe inflammatory lesions were observed in the LF82N + DSS mice when compared to LF82A + DSS mice, and were characterized as transmural in most of the LF82N + DSS mice. Colitis was accompanied by secretion of proinflammatory cytokines (IFNγ, IL-17) and specific mRNA transcripts within the colonic mucosa. Using 16S rRNA gene amplicon sequencing, LF82 colonization did not induce significant changes in the ASF community; however, minimal changes in spatial redistribution by fluorescent in situ hybridization were observed. These results suggest that the age at which mice were colonized with E. coli LF82 pathobiont differentially impacted severity of subsequent colitic events.

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.

Rules of Engagement: Epithelial-Microbe Interactions and Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex, multifactorial disorders that lead to chronic and relapsing intestinal inflammation. The exact etiology remains unknown, however multiple factors including the environment, genetic, dietary, mucosal immunity, and altered microbiome structure and function play important roles in disease onset and progression. Supporting this notion that the gut microbiota plays a pivotal role in IBD pathogenesis, studies in gnotobiotic mice have shown that mouse models of intestinal inflammation require a microbial community to develop colitis. Additionally, antimicrobial therapy in some IBD patients will temporarily induce remission further demonstrating an association between gut microbes and intestinal inflammation. Finally, a dysfunctional intestinal epithelial barrier is also recognized as a key pathogenic factor in IBD. The intestinal epithelium serves as a barrier between the luminal environment and the mucosal immune system and guards against harmful molecules and microorganisms while being permeable to essential nutrients and solutes. Beneficial (i.e., mutualists) bacteria promote mucosal health by strengthening barrier integrity, increasing local defenses (mucin and IgA production) and inhibiting pro-inflammatory immune responses and apoptosis to promote mucosal homeostasis. In contrast, pathogenic bacteria and pathobionts suppress expression and localization of tight junction proteins, cause dysregulation of apoptosis/proliferation and increase pro-inflammatory signaling that directly damages the intestinal mucosa. This review article will focus on the role of intestinal epithelial cells (IECs) and the luminal environment acting as mediators of barrier function in IBD. We will also share some of our translational observations of interactions between IECs, immune cells, and environmental factors contributing to maintenance of mucosal homeostasis, as it relates to GI inflammation and IBD in different animal models.

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.

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.

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4ß.

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 MUC4ß-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 MUC4ß 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 MUC4ß-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4ß-loaded nanoparticles generated MUC4ß-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4ß vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Polymeric Nanoparticle-Based Vaccine Adjuvants and Delivery Vehicles.

As vaccine formulations have progressed from including live or attenuated strains of pathogenic components for enhanced safety, developing new adjuvants to more effectively generate adaptive immune responses has become necessary. In this context, polymeric nanoparticles have emerged as a promising platform with multiple advantages, including the dual capability of adjuvant and delivery vehicle, administration via multiple routes, induction of rapid and long-lived immunity, greater shelf-life at elevated temperatures, and enhanced patient compliance. This comprehensive review describes advances in nanoparticle-based vaccines (i.e., nanovaccines) with a particular focus on polymeric particles as adjuvants and delivery vehicles. Examples of the nanovaccine approach in respiratory infections, biodefense, and cancer are discussed.

Campylobacter jejuni persistently colonizes gnotobiotic altered Schaedler flora C3H/HeN mice and induces mild colitis.

Campylobacter jejuni is a major cause of food-borne human bacterial gastroenteritis but animal models for C. jejuni mediated disease remain limited because C. jejuni poorly colonizes immunocompetent, conventionally-reared (Conv-R) mice. Thus, a reliable rodent model (i.e. persistent colonization) is desirable in order to evaluate C. jejuni-mediated gastrointestinal disease and mechanisms of pathogenicity. As the nature and complexity of the microbiota likely impacts colonization resistance for C. jejuni, Conv-R and gnotobiotic C3H/HeN mice were used to evaluate the persistence of C. jejuni colonization and development of disease. A total of four C. jejuni isolates readily and persistently colonized ASF mice and induced mild mucosal inflammation in the proximal colon, but C. jejuni did not stably colonize nor induce lesions in Conv-R mice. This suggests that the pathogenesis of C. jejuni is influenced by the microbiota, and that ASF mice offer a reproducible model to study the influence of the microbiota on the ability of C. jejuni to colonize the gut and to mediate gastroenteritis.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.

Temporal Dynamics of Chronic Inflammation on the Cecal Microbiota in IL-10-/- Mice.

The intestinal microbiota is a critical component of mucosal health as evidenced by the fact that alterations in the taxonomic composition of the gastrointestinal microbiota are associated with inflammatory bowel diseases. To better understand how the progression of inflammation impacts the composition of the gastrointestinal microbiota, we used culture independent taxonomic profiling to identify temporal changes in the cecal microbiota of C3Bir IL-10-/- mice concomitantly with the onset and progression of colitis. This analysis revealed that IL-10-/- mice displayed a biphasic progression in disease severity, as evidenced by histopathological scores and cytokine production. Beginning at 4 weeks of age, pro-inflammatory cytokines including TNF-α, IFN-γ, IL-6, G-CSF, and IL-1α as well as chemokines including RANTES and MIP-1α were elevated in the serum of IL-10-/- mice. By 19 weeks of age, the mice developed clinical signs of disease as evidenced by weight loss, which was accompanied by a significant increase in serum levels of KC and IL-17. While the overall diversity of the microbiota of both wild type and IL-10-/- were similar in young mice, the latter failed to increase in complexity as the mice matured and experienced changes in abundance of specific bacterial taxa that are associated with inflammatory bowel disease in humans. Collectively, these results reveal that there is a critical time in young mice between four to six weeks of age when inflammation and the associated immune responses adversely affect maturation of the microbiota.

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.

Amphiphilic polyanhydride-based recombinant MUC4ß-nanovaccine activates dendritic cells.

Mucin 4 (MUC4) is a high molecular weight glycoprotein that is differentially overexpressed in pancreatic cancer (PC), functionally contributes to disease progression, and correlates with poor survival. Further, due to its aberrant glycosylation and extensive splicing, MUC4 is a potential target for cancer immunotherapy. Our previous studies have demonstrated the utility of amphiphilic polyanhydride nanoparticles as a useful platform for the development of protein-based prophylactic and therapeutic vaccines. In the present study, we encapsulated purified recombinant human MUC4-beta (MUC4ß) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4ß-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4ß-nanovaccine exhibited significant increase in the surface expressions of MHC I and MHC II and costimulatory molecules (CD80 and CD86), as well as, secretion of pro-inflammatory cytokines (IFN-γ, IL-6, and IL-12) as compared to cells exposed to MUC4ß alone or MUC4ß mixed with blank nanoparticles (MUC4ß+NP). Following immunization, as compared to the other formulations, MUC4ß-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4ß-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4ß-nanovaccine as a novel platform for PC immunotherapy.