Publications by authors named "Balaji Narasimhan"

124 Publications

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

J Vis Exp 2021 Jun 28(172). Epub 2021 Jun 28.

Interdisciplinary Graduate Program in Human Toxicology, University of Iowa; Department of Pathology, University of Iowa; Department of Chemical and Biological Engineering, College of Engineering, Iowa State University; Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa; Holden Comprehensive Cancer Center, University of Iowa; Department of Oral Pathology, Radiology and Medicine, College of Dentistry, University of Iowa;

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.
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http://dx.doi.org/10.3791/62683DOI Listing
June 2021

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

Vaccine 2021 06 2;39(29):3862-3870. Epub 2021 Jun 2.

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, United States; Nanovaccine Institute, Ames, IA, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vaccine.2021.05.077DOI Listing
June 2021

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

J Immunol 2021 May 7;206(9):2122-2134. Epub 2021 Apr 7.

Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA;

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in both young children and in older adults. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is currently available. We have developed a novel RSV vaccine composed of a prefusion-stabilized variant of the fusion (F) protein (DS-Cav1) and a CpG oligodeoxynucleotide adjuvant encapsulated within polyanhydride nanoparticles, termed RSVNanoVax. A prime-boost intranasal administration of RSVNanoVax in BALB/c mice significantly alleviated weight loss and pulmonary dysfunction in response to an RSV challenge, with protection maintained up to at least 6 mo postvaccination. In addition, vaccinated mice exhibited rapid viral clearance in the lungs as early as 2 d after RSV infection in both inbred and outbred populations. Vaccination induced tissue-resident memory CD4 and CD8 T cells in the lungs, as well as RSV F-directed neutralizing Abs. Based on the robust immune response elicited and the high level of durable protection observed, our prefusion RSV F nanovaccine is a promising new RSV vaccine candidate.
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http://dx.doi.org/10.4049/jimmunol.2100018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062305PMC
May 2021

A single dose polyanhydride-based nanovaccine against paratuberculosis infection.

NPJ Vaccines 2020 Feb 14;5(1):15. Epub 2020 Feb 14.

Department of Pathobiological Sciences, University of Wisconsin, Madison, WI, 53706, USA.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) causes Johne's disease in ruminants and is characterized by chronic gastroenteritis leading to heavy economic losses to the dairy industry worldwide. The currently available vaccine (inactivated bacterin in oil base) is not effective in preventing pathogen shedding and is rarely used to control Johne's disease in dairy herds. To develop a better vaccine that can prevent the spread of Johne's disease, we utilized polyanhydride nanoparticles (PAN) to encapsulate mycobacterial antigens composed of whole cell lysate (PAN-Lysate) and culture filtrate (PAN-Cf) of M. paratuberculosis. These nanoparticle-based vaccines (i.e., nanovaccines) were well tolerated in mice causing no inflammatory lesions at the site of injection. Immunological assays demonstrated a substantial increase in the levels of antigen-specific T cell responses post-vaccination in the PAN-Cf vaccinated group as indicated by high percentages of triple cytokine (IFN-γ, IL-2, TNF-α) producing CD8 T cells. Following challenge, animals vaccinated with PAN-Cf continued to produce significant levels of double (IFN-γ, TNF-α) and single cytokine (IFN-γ) secreting CD8 T cells compared with animals vaccinated with an inactivated vaccine. A significant reduction in bacterial load was observed in multiple organs of animals vaccinated with PAN-Cf, which is a clear indication of protection. Overall, the use of polyanhydride nanovaccines resulted in development of protective and sustained immunity against Johne's disease, an approach that could be applied to counter other intracellular pathogens.
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http://dx.doi.org/10.1038/s41541-020-0164-yDOI Listing
February 2020

Synthesis and Characterization of Rapidly Degrading Polyanhydrides as Vaccine Adjuvants.

ACS Biomater Sci Eng 2020 01 3;6(1):265-276. Epub 2020 Jan 3.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States.

There is a currently a need to develop adjuvants that are best suited to simultaneously enhance immune responses, induce immunologic memory, improve patient compliance (i.e., reduce doses and inflammation), and provide vaccine shelf stability for stockpiling and global deployment to challenging environments. Biodegradable polyanhydrides have been investigated extensively to overcome such challenges. It has been shown that controlling copolymer composition can result in chemistry-dependent immunomodulatory capabilities. These studies have revealed that copolymers rich in sebacic acid (SA) are highly internalized by antigen presenting cells and confer improved shelf stability of encapsulated proteins, while copolymers rich in 1,8-bis(-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) also exhibit enhanced internalization by and activation of antigen presenting cells (APCs), in addition to providing superior retention of protein stability following encapsulation and release. However, to date, CPTEG:SA copolymers have not been synthesized and described. In this work, we hypothesized that new copolymers composed of CPTEG and SA would combine the advantages of both monomers in terms of enhanced thermal properties, maintaining antigenicity of encapsulated proteins following nanoparticle synthesis, and superior cellular internalization and activation by APCs, demonstrated by the upregulation of costimulatory markers CD80, CD86, and CD40, as well as the secretion of proinflammatory cytokines IL-6, IL-1β, and TNF-α. Herein, we describe the synthesis and design of novel CPTEG:SA nanoparticles with improved thermal properties, payload stability, and internalization by antigen presenting cells for applications in vaccine delivery. The performance of these new CPTEG:SA formulations was compared to that of traditional polyanhydride copolymers.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01427DOI Listing
January 2020

Applications of Nanovaccines for Disease Prevention in Cattle.

Front Bioeng Biotechnol 2020 11;8:608050. Epub 2020 Dec 11.

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, United States.

Vaccines are one of the most important tools available to prevent and reduce the incidence of infectious diseases in cattle. Despite their availability and widespread use to combat many important pathogens impacting cattle, several of these products demonstrate variable efficacy and safety in the field, require multiple doses, or are unstable under field conditions. Recently, nanoparticle-based vaccine platforms (nanovaccines) have emerged as promising alternatives to more traditional vaccine platforms. In particular, polymer-based nanovaccines provide sustained release of antigen payloads, stabilize such payloads, and induce enhanced antibod- and cell-mediated immune responses, both systemically and locally. To improve vaccine administrative strategies and efficacy, they can be formulated to contain multiple antigenic payloads and have the ability to protect fragile proteins from degradation. Nanovaccines are also stable at room temperature, minimizing the need for cold chain storage. Nanoparticle platforms can be synthesized for targeted delivery through intranasal, aerosol, or oral administration to induce desired mucosal immunity. In recent years, several nanovaccine platforms have emerged, based on biodegradable and biocompatible polymers, liposomes, and virus-like particles. While most nanovaccine candidates have not yet advanced beyond testing in rodent models, a growing number have shown promise for use against cattle infectious diseases. This review will highlight recent advancements in polymeric nanovaccine development and the mechanisms by which nanovaccines may interact with the bovine immune system. We will also discuss the positive implications of nanovaccines use for combating several important viral and bacterial disease syndromes and consider important future directions for nanovaccine development in beef and dairy cattle.
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http://dx.doi.org/10.3389/fbioe.2020.608050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759628PMC
December 2020

Enzyme Immunoassay-Based Platform for Accurate Detection of Serum Pathological α-Synuclein in Parkinson's Disease Patients.

ACS Chem Neurosci 2020 12 16;11(24):4179-4190. Epub 2020 Nov 16.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States.

An assay for accurately diagnosing early stage Parkinson's Disease (PD) is currently unavailable, and therefore, there is an urgent and unmet need. Such a diagnostic assay will enable prompt institution of appropriate supportive management measures to prevent rapid deterioration of disease and improve both quality of life and life expectancy of PD patients. A reliable assay platform will also be of great benefit to drug discovery and drug development in the area of PD. To this end, we describe the development of two indirect, competitive, semiquantitative enzyme immunoassays (EIAs), each employing a disparate singularly specific mouse monoclonal antibody (ssMAb) against pathological aggregates of human α-Synuclein (αSyn), a well-established biomarker pathognomonic of PD. Our results demonstrate that these EIAs in tandem accurately discriminated between αSyn serum concentrations from PD patients and age-matched healthy control (HC) individuals (PD = 1700 ± 220 ng/mL; HC = 870 ± 120 ng/mL with an overall sensitivity of 56%, specificity of 63%, positive predictive value of 60%, and negative predictive value of 59%). The limits of detection of αSyn were 400 and 300 pg/mL for ssMAbs 3C5 and 5H6, respectively. These tandem EIAs have the potential to add to the repertoire of tools for earlier diagnosis of this debilitating disorder, as well as for drug development strategies.
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http://dx.doi.org/10.1021/acschemneuro.0c00461DOI Listing
December 2020

Polymeric Nanoparticle-Based Vaccine Adjuvants and Delivery Vehicles.

Curr Top Microbiol Immunol 2020 Nov 10. Epub 2020 Nov 10.

Departments of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, USA.

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.
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http://dx.doi.org/10.1007/82_2020_226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107186PMC
November 2020

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

Int J Nanomedicine 2020 7;15:6579-6592. Epub 2020 Sep 7.

Department of Veterinary Microbiology and Preventative Medicine, Iowa State University, Ames, IA, USA.

Introduction: Adjuvants and immunotherapies designed to activate adaptive immunity to eliminate infectious disease and tumors have become an area of interest aimed at providing a safe and effective strategy to prevent or eliminate disease. Existing approaches would benefit from the development of immunization regimens capable of inducing efficacious cell-mediated immunity directed toward CD8 T cell-specific antigens. This goal is critically dependent upon appropriate activation of antigen-presenting cells (APCs) most notably dendritic cells (DCs). In this regard, polyanhydride particles have been shown to be effectively internalized by APCs and induce activation.

Methods: Here, a prophylactic vaccine regimen designed as a single-dose polyanhydride nanovaccine encapsulating antigen is evaluated for the induction of CD8 T cell memory in a model system where antigen-specific protection is restricted to CD8 T cells. Bone marrow-derived dendritic cells (BMDCs) are used as an in vitro model system to evaluate the magnitude and phenotype of APC activation. Primary DCs, particularly those with described ability to activate CD8 T cells, are also evaluated for their in vitro responses to polyanhydride nanoparticles.

Results: Herein, polyanhydride nanoparticles are shown to induce potent in vitro upregulation of costimulatory molecules on the cell surface of BMDCs. In contrast to the classically used TLR agonists, nanoparticles did not induce large amounts of pro-inflammatory cytokines, did not induce characteristic metabolic response of DCs, nor produce innate antimicrobial effector molecules, such as nitric oxide (NO). The polyanhydride nanovaccine results in protective CD8 T cell responses as measured by inhibition of tumor progression and survival.

Discussion: Together, these results suggest that the use of a polyanhydride-based nanovaccine can be an effective approach to inducing antigen-specific CD8 T cell memory by providing antigen delivery and DC activation while avoiding overt inflammatory responses typically associated with traditional adjuvants.
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http://dx.doi.org/10.2147/IJN.S261041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490050PMC
December 2020

Biodistribution of degradable polyanhydride particles in Aedes aegypti tissues.

PLoS Negl Trop Dis 2020 09 8;14(9):e0008365. Epub 2020 Sep 8.

Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin, United States of America.

Insecticide resistance poses a significant threat to the control of arthropods that transmit disease agents. Nanoparticle carriers offer exciting opportunities to expand the armamentarium of insecticides available for public health and other pests. Most chemical insecticides are delivered by contact or feeding, and from there must penetrate various biological membranes to reach target organs and kill the pest organism. Nanoparticles have been shown to improve bioactive compound navigation of such barriers in vertebrates, but have not been well-explored in arthropods. In this study, we explored the potential of polyanhydride micro- and nanoparticles (250 nm- 3 μm), labeled with rhodamine B to associate with and/or transit across insect biological barriers, including the cuticle, epithelium, midgut and ovaries, in female Ae. aeygpti mosquitoes. Mosquitoes were exposed using conditions to mimic surface contact with a residual spray or paint, topical exposure to mimic contact with aerosolized insecticide, or per os in a sugar meal. In surface contact experiments, microparticles were sometimes observed in association with the exterior of the insect cuticle. Nanoparticles were more uniformly distributed across exterior tissues and present at higher concentrations. Furthermore, by surface contact, topical exposure, or per os, particles were detected in internal organs. In every experiment, amphiphilic polyanhydride nanoparticles associated with internal tissues to a higher degree than hydrophobic nanoparticles. In vitro, nanoparticles associated with Aedes aegypti Aag2 cells within two hours of exposure, and particles were evident in the cytoplasm. Further studies demonstrated that particle uptake is dependent on caveolae-mediated endocytosis. The propensity of these nanoparticles to cross biological barriers including the cuticle, to localize in target tissue sites of interest, and to reach the cytoplasm of cells, provides great promise for targeted delivery of insecticidal candidates that cannot otherwise reach these cellular and subcellular locations.
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http://dx.doi.org/10.1371/journal.pntd.0008365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500644PMC
September 2020

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4β.

J Biomed Mater Res A 2021 Jun 25;109(6):893-902. Epub 2020 Aug 25.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, USA.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.
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http://dx.doi.org/10.1002/jbm.a.37080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100985PMC
June 2021

Chitosan-adjuvanted Salmonella subunit nanoparticle vaccine for poultry delivered through drinking water and feed.

Carbohydr Polym 2020 Sep 19;243:116434. Epub 2020 May 19.

Food Animal Health Research Program, Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, OH, 44691, USA; Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, 43210, USA. Electronic address:

Poor induction of mucosal immunity in the intestines by current Salmonella vaccines is a challenge to the poultry industry. We prepared and tested an oral deliverable Salmonella subunit vaccine containing immunogenic outer membrane proteins (OMPs) and flagellin (F) protein loaded and F-protein surface coated chitosan nanoparticles (CS NPs) (OMPs-F-CS NPs). The OMPs-F-CS NPs had mean particle size distribution of 514 nm, high positive charge and spherical in shape. In vitro and in vivo studies revealed the F-protein surface coated CS NPs were specifically targeted to chicken immune cells. The OMPs-F-CS NPs treatment of chicken immune cells upregulated TLRs, and Th1 and Th2 cytokines mRNA expression. Oral delivery of OMPs-F-CS NPs in birds enhanced the specific systemic IgY and mucosal IgA antibodies responses as well as reduced the challenge Salmonella load in the intestines. Thus, user friendly oral deliverable chitosan-based Salmonella vaccine for poultry is a viable alternative to current vaccines.
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http://dx.doi.org/10.1016/j.carbpol.2020.116434DOI Listing
September 2020

A single dose polyanhydride-based nanovaccine against paratuberculosis infection.

NPJ Vaccines 2020 14;5:15. Epub 2020 Feb 14.

1Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706 USA.

subsp () causes Johne's disease in ruminants and is characterized by chronic gastroenteritis leading to heavy economic losses to the dairy industry worldwide. The currently available vaccine (inactivated bacterin in oil base) is not effective in preventing pathogen shedding and is rarely used to control Johne's disease in dairy herds. To develop a better vaccine that can prevent the spread of Johne's disease, we utilized polyanhydride nanoparticles (PAN) to encapsulate mycobacterial antigens composed of whole cell lysate (PAN-Lysate) and culture filtrate (PAN-Cf) of . These nanoparticle-based vaccines (i.e., nanovaccines) were well tolerated in mice causing no inflammatory lesions at the site of injection. Immunological assays demonstrated a substantial increase in the levels of antigen-specific T cell responses post-vaccination in the PAN-Cf vaccinated group as indicated by high percentages of triple cytokine (IFN-γ, IL-2, TNF-α) producing CD8 T cells. Following challenge, animals vaccinated with PAN-Cf continued to produce significant levels of double (IFN-γ, TNF-α) and single cytokine (IFN-γ) secreting CD8 T cells compared with animals vaccinated with an inactivated vaccine. A significant reduction in bacterial load was observed in multiple organs of animals vaccinated with PAN-Cf, which is a clear indication of protection. Overall, the use of polyanhydride nanovaccines resulted in development of protective and sustained immunity against Johne's disease, an approach that could be applied to counter other intracellular pathogens.
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http://dx.doi.org/10.1038/s41541-020-0164-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021715PMC
February 2020

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

ACS Comb Sci 2020 04 11;22(4):172-183. Epub 2020 Mar 11.

Department of Chemical and Biological Engineering and Nanovaccine Institute, Iowa State University, Ames, Iowa 50011, United States.

Combinatorial techniques can accelerate the discovery and development of polymeric nanodelivery devices by pairing high-throughput synthesis with rapid materials characterization. Biodegradable polyanhydrides demonstrate tunable release, high cellular internalization, and dose sparing properties when used as nanodelivery devices. This nanoparticle platform shows promising potential for small molecule drug delivery, but the pace of understanding and rational design of these nanomedicines is limited by the low throughput of conventional characterization. This study reports the use of a high-throughput method to synthesize libraries of a newly synthesized, rapidly eroding polyanhydride copolymer based on 1,8-bis(-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and sebacic acid (SA) monomers. The high-throughput method enabled efficient screening of copolymer microstructure, revealing weak block-type and alternating architectures. The high-throughput method was adapted to synthesize nanoparticle libraries encapsulating hydrophobic model drugs. Drug release from these nanoparticles was rapid, with a majority of the payload released within 3 days. Drug release was dramatically slowed at acidic pH, which could be useful for oral drug delivery. Rhodamine B (RhoB) release kinetics generally followed patterns of polymer erosion kinetics, while Coomassie brilliant blue (CBB) released the fastest from the slowest degrading polymer chemistry and vice versa. These differences in trends between copolymer chemistry and release kinetics were hypothesized to arise from differences in mixing thermodynamics. A high-throughput method was developed to synthesize polymer-drug film libraries and characterize mixing thermodynamics by melting point depression. Rhodamine B had a negative χ for all copolymers with <30 mol % CPTEG tested, indicating a tendency toward miscibility. By contrast, CBB χ increased, eventually becoming positive near 15:85 CPTEG:SA, with increasing CPTEG content. This indicates an increasing tendency toward phase separation in CPTEG-rich copolymers. These results screening polymer-drug interactions showed good agreement with predictions from Hansen solubility parameter estimation and were able to explain the observed differences in model drug release trends.
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http://dx.doi.org/10.1021/acscombsci.9b00162DOI Listing
April 2020

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

J Anim Sci 2020 06;98(6)

Department of Anatomy and Physiology, Kansas State University, Manhattan, KS.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.
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http://dx.doi.org/10.1093/jas/skz392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271671PMC
June 2020

Vitamin A deficiency impairs the immune response to intranasal vaccination and RSV infection in neonatal calves.

Sci Rep 2019 10 22;9(1):15157. Epub 2019 Oct 22.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA, USA.

Respiratory syncytial virus (RSV) infection is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Vitamin A deficiency (VAD) is one of the most prevalent nutrition-related health problems in the world and is a significant risk factor in the development of severe respiratory infections in infants and young children. Bovine RSV (BRSV) is a primary cause of lower respiratory tract disease in young cattle. The calf model of BRSV infection is useful to understand the immune response to human RSV infection. We have previously developed an amphiphilic polyanhydride nanoparticle (NP)-based vaccine (i.e., nanovaccine) encapsulating the fusion and attachment proteins from BRSV (BRSV-NP). Calves receiving a single, intranasal dose of the BRSV-NP vaccine are partially protected from BRSV challenge. Here, we evaluated the impact of VAD on the immune response to the BRSV-NP vaccine and subsequent challenge with BRSV. Our results show that VAD calves are unable to respond to the mucosal BRSV-NP vaccine, are afforded no protection from BRSV challenge and have significant abnormalities in the inflammatory response in the infected lung. We further show that acute BRSV infection negatively impacts serum and liver retinol, rendering even well-nourished individuals susceptible to VAD. Our results support the use of the calf model for elucidating the impact of nutritional status on mucosal immunity and respiratory viral infection in infants and underline the importance of VA in regulating immunity in the respiratory mucosa.
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http://dx.doi.org/10.1038/s41598-019-51684-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805856PMC
October 2019

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

Acta Biomater 2019 12 11;100:326-337. Epub 2019 Oct 11.

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, United States; Nanovaccine Institute, Iowa State University, Ames, IA, United States. Electronic address:

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-R,R-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.
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http://dx.doi.org/10.1016/j.actbio.2019.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012387PMC
December 2019

Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis.

Heliyon 2019 Aug 29;5(8):e02370. Epub 2019 Aug 29.

Infectious Bacterial Diseases Research Unit, National Animal Disease Centers, United States Department of Agriculture, 1920 Dayton Avenue, Ames, IA, 50010, USA.

Brucellosis is a bacterial zoonosis and a significant source of economic loss and a major public health concern, worldwide. Bovine brucellosis, as caused primarily by , is an important cause of reproductive loss in cattle. Vaccination has been the most effective way to reduce disease prevalence contributing to the success of control and eradication programs. Currently, there are no human vaccines available, and despite the success of commercial vaccines for livestock, such as strain RB51 (RB51), there is need for development of novel and safer vaccines against brucellosis. In the current study, we report the fabrication of and immune responses to an implantable single dose polyanhydride-based, methanol-killed RB51 antigen containing delivery platform (VPEAR) in cattle. In contrast to animals vaccinated with RB51, we did not observe measurable RB51-specific IFN-γ or IgG responses in the peripheral blood, following initial vaccination with VPEAR. However, following a subsequent booster vaccination with RB51, we observed an anamnestic response in both vaccination treatments (VPEAR and live RB51). The magnitude and kinetics of CD4+ IFN-γ-mediated responses and circulating memory T cell subpopulations were comparable between the two vaccination treatments. Additionally, IgG titers were significantly increased in animals vaccinated with VPEAR as compared to live RB51- vaccinated animals. These data demonstrate that killed antigen may be utilized to generate and sustain memory, IFN-γ-mediated, CD4+ T cell and humoral responses against Brucella in a natural host. To our knowledge, this novel approach to vaccination against intracellular bacteria, such as , has not been reported before.
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http://dx.doi.org/10.1016/j.heliyon.2019.e02370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728543PMC
August 2019

Pentaerythritol-based lipid A bolsters the antitumor efficacy of a polyanhydride particle-based cancer vaccine.

Nanomedicine 2019 10 15;21:102055. Epub 2019 Jul 15.

Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, USA; Nanovaccine Institute, Iowa State University, Ames, IA and University of Iowa, Iowa City, IA, USA. Electronic address:

The primary objective of this study was to enhance the antitumor efficacy of a model cancer vaccine through co-delivery of pentaerythritol lipid A (PELA), an immunological adjuvant, and a model tumor antigen, ovalbumin (OVA), separately loaded into polyanhydride particles (PA). In vitro experiments showed that encapsulation of PELA into PA (PA-PELA) significantly enhanced its stimulatory capacity on dendritic cells as evidenced by increased levels of the cell surface costimulatory molecules, CD80/CD86. In vivo experiments showed that PA-PELA, in combination with OVA-loaded PA (PA-OVA), significantly expanded the OVA-specific CD8 T lymphocyte population compared to PA-OVA alone. Furthermore, OVA-specific serum antibody titers of mice vaccinated with PA-OVA/PA-PELA displayed a significantly stronger shift toward a Th1-biased immune response compared to PA-OVA alone, as evidenced by the substantially higher IgG:IgG ratios achieved by the former. Analysis of E.G7-OVA tumor growth curves showed that mice vaccinated with PA-OVA/PA-PELA had the slowest average tumor growth rate.
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http://dx.doi.org/10.1016/j.nano.2019.102055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814548PMC
October 2019

Effective mosaic-based nanovaccines against avian influenza in poultry.

Vaccine 2019 08 9;37(35):5051-5058. Epub 2019 Jul 9.

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA; Pan Genome Systems, Madison, WI, USA. Electronic address:

Avian influenza virus (AIV) is an extraordinarily diverse pathogen that causes significant morbidity in domesticated poultry populations and threatens human life with looming pandemic potential. Controlling avian influenza in susceptible populations requires highly effective, economical and broadly reactive vaccines. Several AIV vaccines have proven insufficient despite their wide use, and better technologies are needed to improve their immunogenicity and broaden effectiveness. Previously, we developed a "mosaic" H5 subtype hemagglutinin (HA) AIV vaccine and demonstrated its broad protection against diverse highly pathogenic H5N1 and seasonal H1N1 virus strains in mouse and non-human primate models. There is a significant interest in developing effective and safe vaccines against AIV that cannot contribute to the emergence of new strains of the virus once circulating in poultry. Here, we report on the development of an H5 mosaic (H5M) vaccine antigen formulated with polyanhydride nanoparticles (PAN) that provide sustained release of encapsulated antigens. H5M vaccine constructs were immunogenic whether delivered by the modified virus Ankara (MVA) strain or encapsulated within PAN. Both humoral and cellular immune responses were generated in both specific-pathogen free (SPF) and commercial chicks. Importantly, chicks vaccinated by H5M constructs were protected in terms of viral shedding from divergent challenge with a low pathogenicity avian influenza (LPAI) strain at 8 weeks post-vaccination. In addition, protective levels of humoral immunity were generated against highly pathogenic avian influenza (HPAI) of the similar H5N1 and genetically dissimilar H5N2 viruses. Overall, the developed platform technologies (MVA vector and PAN encapsulation) were safe and provided high levels of sustained protection against AIV in chickens. Such approaches could be used to design more efficacious vaccines against other important poultry infections.
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http://dx.doi.org/10.1016/j.vaccine.2019.06.077DOI Listing
August 2019

Amphiphilic polyanhydride-based recombinant MUC4β-nanovaccine activates dendritic cells.

Genes Cancer 2019 May;10(3-4):52-62

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

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 (MUC4β) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4β-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4β-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 MUC4β alone or MUC4β mixed with blank nanoparticles (MUC4β+NP). Following immunization, as compared to the other formulations, MUC4β-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4β-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4β-nanovaccine as a novel platform for PC immunotherapy.
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http://dx.doi.org/10.18632/genesandcancer.189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584211PMC
May 2019

STING pathway stimulation results in a differentially activated innate immune phenotype associated with low nitric oxide and enhanced antibody titers in young and aged mice.

Vaccine 2019 05 12;37(20):2721-2730. Epub 2019 Apr 12.

Iowa State University, Department of Veterinary Microbiology and Preventative Medicine, United States; Nanovaccine Institute, Iowa State University, United States. Electronic address:

Background: One of the most concerning public health issues, related to vaccination and disease prevention, is the inability to induce durable immune responses following a single-dose immunization. In this regard, the nature of the inflammatory environment induced by vaccine adjuvants can negatively impact the resulting immune response. To address these concerns, new strategies to vaccine design are needed in order to improve the outcomes of immune responses, particularly in immunologically disadvantaged populations.

Methods: Comparisons of the scope of innate immune activation induced by TLR agonists versus cyclic dinucleotides (CDNs) was performed. Their effects on the activation characteristics (e.g., metabolism, cytokine secretion) of bone marrow derived dendritic cells (BMDCs) were studied. In addition, the differential effects on in vivo induction of antibody responses were measured.

Results: As compared to TLR ligands, the stimulation of BMDCs with CDNs induced distinctly different metabolic outcomes. Marked differences were observed in the production of nitric oxide (NO) and the cytokine BAFF. These distinct differences were correlated with improved (i.e., more rapid and persistent) vaccine antibody responses in both aged and young mice.

Conclusions: Our results illustrate that the innate immune pathway targeted by adjuvants can critically impact the outcome of the immune response post-vaccination. Specifically, CDN stimulation of APCs induced an activation phenotype that was characterized by decreased innate effector molecule production (e.g., NO) and increased BAFF. This was attributed to the induction of an innate inflammatory environment that enabled the host to make the most of the existing B lymphocyte potential. The use of adjuvants that differentially engage mechanisms of innate immune activation would be particularly advantageous for the generation of robust, single dose vaccines. The results of this study demonstrated that CDNs induced differential innate activation and enhanced vaccine induced antibody responses in both young and aged mice.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499688PMC
May 2019

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

Mol Pharm 2019 05 26;16(5):1917-1928. Epub 2019 Apr 26.

Department of Materials Design and Innovation , University at Buffalo , Buffalo , New York 14260 , United States.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b01272DOI Listing
May 2019

Safety and biocompatibility of injectable vaccine adjuvants composed of thermogelling block copolymer gels.

J Biomed Mater Res A 2019 08 29;107(8):1754-1762. Epub 2019 Apr 29.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, 50011.

Injectable thermogelling polymers have been recently investigated as novel adjuvants and delivery systems for next generation vaccines. As research into natural and synthetic biocompatible polymers progresses, the safety and biocompatibility of these compounds is of paramount importance. We have developed cationic pentablock copolymer (PBC) vaccine adjuvants based on Pluronic F127, a thermogelling triblock copolymer that has been approved by the FDA for multiple applications, and methacrylated poly(diethyl amino)ethyl methacrylate outer blocks. These novel materials have been demonstrated to effectively create an antigen depot, minimally impact antigen stability, and enhance the immune response to antigens (i.e., adjuvanticity) in mice. In this work, we investigated the safety and biocompatibility of the parent triblock Pluronic gels and the cationic PBC gels in mice. Histological analysis showed no injection site reactions and no damage to the liver or kidneys was observed upon administering the block copolymer formulations. However, the subcutaneous injection of a thermogelling Pluronic solution induced increased levels of lipids in the blood, with no further deleterious effects observed from the addition of the cationic outer blocks. This hyperlipidemia resolved within 30 days after the administration of the Pluronic formulation. To mitigate this adverse effect, the vaccine adjuvant formulations were modified by adding poly(vinyl alcohol), which allowed gelation, while reducing the amount of Pluronic in the formulation. This modified formulation abrogated the observed hyperlipidemia and no adverse effects were observed in the serum through biomarker analysis or at the injection site (i.e., inflammation) in comparison to the responses induced by administration of saline or incomplete Freund's adjuvant. These studies provide a foundation to developing these gels as adjuvants for next generation vaccines. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1754-1762, 2019.
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http://dx.doi.org/10.1002/jbm.a.36691DOI Listing
August 2019

Design and synthesis of multivalent α-1,2-trimannose-linked bioerodible microparticles for applications in immune response studies of infection.

Beilstein J Org Chem 2019 11;15:623-632. Epub 2019 Mar 11.

Department of Chemistry, Indiana University Bloomington, 800 E. Kirkwood Ave., Bloomington, Indiana 47405-7102, USA.

Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new cases each year in predominately underdeveloped countries. The treatment of the disease is severely underdeveloped due to the ability of the pathogen to evade and abate immune responses. In an effort to develop anti-leishmaniasis vaccines and adjuvants, novel carbohydrate-based probes were made to study the mechanisms of immune modulation. In this study, a new bioerodible polyanhydride microparticle was designed and conjugated with a glycodendrimer molecular probe. This molecular probe incorporates a pathogen-like multivalent display of α-1,2-trimannose, for which a more efficient synthesis was designed, with a tethered fluorophore. Further attachment of the glycodendrimer to a biocompatible, surface eroding microparticle allows for targeted uptake and internalization of the pathogen-associated oligosaccharide by phagocytic immune cells. The α-1,2-trimannose-linked bioerodible microparticles were found to be safe after administration into the footpad of mice and demonstrated a similar response to α-1,2-trimannose-coated latex beads during footpad infection. Furthermore, the bioerodible microparticles allowed for investigation of the role of pathogen-associated oligosaccharides for recognition by pathogen-recognition receptors during induced leishmaniasis.
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http://dx.doi.org/10.3762/bjoc.15.58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423605PMC
March 2019

Interleukin-1 alpha increases anti-tumor efficacy of cetuximab in head and neck squamous cell carcinoma.

J Immunother Cancer 2019 03 19;7(1):79. Epub 2019 Mar 19.

Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA, 52242, USA.

Background: Despite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab.

Methods: Parental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4 and CD8 T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS).

Results: Cetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels.

Conclusions: Altogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs.
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http://dx.doi.org/10.1186/s40425-019-0550-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425573PMC
March 2019

Pentablock Copolymer Micelle Nanoadjuvants Enhance Cytosolic Delivery of Antigen and Improve Vaccine Efficacy while Inducing Low Inflammation.

ACS Biomater Sci Eng 2019 Mar 18;5(3):1332-1342. Epub 2019 Feb 18.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States.

As the focus has shifted from traditional killed or live, attenuated vaccines toward subunit vaccines, improvements in vaccine safety have been confronted with low immunogenicity of protein antigens. This issue has been addressed by synthesizing and designing a wide variety of antigen carriers and adjuvants, such as Toll-like receptor agonists (e.g., MPLA, CpG). Studies have focused on optimizing adjuvants for improved cellular trafficking, cytosolic availability, and improved antigen presentation. In this work, we describe the design of novel amphiphilic pentablock copolymer (PBC) adjuvants that exhibit high biocompatibility and reversible pH- and temperature-sensitive micelle formation. We demonstrate improved humoral immunity in mice in response to single-dose immunization with PBC micelle adjuvants compared with soluble antigen alone. With the motive of exploring the mechanism of action of these PBC micelles, we studied intracellular trafficking of these PBC micelles with a model antigen and demonstrated that the PBC micelles associate with the antigen and enhance its cytosolic delivery to antigen-presenting cells. We posit that these PBC micelles operate via immune-enhancing mechanisms that are different from that of traditional Toll-like receptor activating adjuvants. The metabolic profile of antigen-presenting cells stimulated with traditional adjuvants and the PBC micelles also suggests distinct mechanisms of action. A key finding from this study is the low production of nitric oxide and reactive oxygen species by antigen-presenting cells when stimulated by PBC micelle adjuvants in sharp contrast to TLR adjuvants. Together, these studies provide a basis for rationally developing novel vaccine adjuvants that are safe, that induce low inflammation, and that can efficiently deliver antigen to the cytosol.
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http://dx.doi.org/10.1021/acsbiomaterials.8b01591DOI Listing
March 2019

Single dose combination nanovaccine provides protection against influenza A virus in young and aged mice.

Biomater Sci 2019 Feb;7(3):809-821

Chemical & Biological Engineering, Iowa State University, Ames, IA 50011, USA.

Immunosenescence poses a formidable challenge in designing effective influenza vaccines for aging populations. While approved vaccines against influenza viruses exist, their efficacy in older adults is significantly decreased due to the diminished capabilities of innate and adaptive immune responses. In this work, the ability of a combination nanovaccine containing both recombinant hemagglutinin and nucleoprotein to provide protection against seasonal influenza virus infection was examined in young and aged mice. Vaccine formulations combining two nanoadjuvants, polyanhydride nanoparticles and pentablock copolymer micelles, were shown to enhance protection against challenge compared to each adjuvant alone in young mice. Nanoparticles were shown to enhance in vitro activation of dendritic cells isolated from aged mice, while both nanoadjuvants did not induce proinflammatory cytokine secretion which may be detrimental in aged individuals. In addition, the combination nanovaccine platform was shown to induce demonstrable antibody titers in both young and aged mice that correlated with the maintenance of body weight post-challenge. Collectively, these data demonstrate that the combination nanovaccine platform is a promising technology for influenza vaccines for older adults.
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http://dx.doi.org/10.1039/c8bm01443dDOI Listing
February 2019

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

J Control Release 2019 01 17;294:288-297. Epub 2018 Dec 17.

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA; Nanovaccine Institute, Iowa State University, Ames, IA 50011, USA; Interdepartmental Microbiology Program, Iowa State University, Ames, IA 50011, USA.

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 log 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.
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http://dx.doi.org/10.1016/j.jconrel.2018.12.024DOI Listing
January 2019