DEVELOPING MORE EFFECTIVE AND SAFE VACCINES WITH NOVEL ADJUVANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013617
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Veterinary Comparative Pathobiology

Non Technical Summary
Infectious diseases continue to cause significant economic losses to production animals. When available, vaccines offer an efficient and cost-effective tool to control infectious diseases. With the emergence of new pathogens such as porcine epidemic diarrhea virus (PEDV), increased antimicrobial resistance, and increased societal pressure to reduce the use of antibiotics, the need for new effective vaccines continues to rise. Several diseases such as influenza not only pose a threat to production animals, but are also zoonotic. Control of these diseases is of importance to public health. Many effective vaccines contain adjuvants, substances that enhance the immune response. Current adjuvants such as aluminum compounds and oil emulsions have drawbacks and there is a need for new adjuvants that can make vaccines more effective while limiting side effects. Moreover, these current adjuvants are not suitable for mucosal delivery of vaccines. The research aims to create novel biodegradable adjuvants that are based on nanoparticles derived from sweet corn. These adjuvants are effective, biodegradable and inexpensive making them excellent candidates for incorporation in animal health vaccines.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3910	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species;

Field Of Science
1090 - Immunology;

Keywords

vaccines

adjuvants

nanoparticles

immune response

influenza

Goals / Objectives
The overall goal of this research is to develop novel adjuvants for injectable and mucosal vaccines that will enhance the efficacy and safety of the vaccines for use in food animals. We recently discovered that chemically modified phytoglycogen nanoparticles derived from sweet corn have immunostimulatory properties and can be used as vaccine adjuvants. The objectives of the proposed research are:To determine the effect of surface charge and hydrophobicity on the interaction with dendritic cells in vitro and the ability to enhance the immune response in vivo.To determine the effect of phytoglycogen nanoparticles on the immune response in a target animal species (swine).To determine the effect of combination of phytoglycogen nanoparticles with other immunostimulatory molecules on the immune response following intramuscular and intranasal immunization.

Project Methods
Objective 1. We have previously shown that phytoglycogen (PG) nanoparticles modified with octenyl succinate (OS) and (3-chloro-2-hydroxypropyl)-trimethylammonium chloride (CHPTAC) have a positive surface charge and act as adjuvants when incorporated in vaccine formulations [1]. Here, we will vary the surface hydrophobicity and charge density by varying the ratio of OS and CHPTAC. The PG nanoparticles will be prepared as described from kernels of a variety of sweet corn, sugary-1 [1,2]. The PG nanoparticles will be mixed with different concentrations of octenyl succinic anhydride followed by CHPTAC to create nine different nanoparticles with different surface charges (0, +9, +18 mV) and three different degrees of hydrophobicity. The size and surface charge nanoparticles will be determined with a zeta-sizer instrument and the hydrophobicity by phase separation (partition coefficient in oil and water). The preparation and physical characterization will be performed by collaborator, Dr. Yuan Yao. The particles will be evaluated for the adsorption of positively and negatively charged proteins. The effect of the nanoparticles on dendritic cells will be performed with primary dendritic cells generated from the bone marrow of mice [1]. The parameters evaluated will include uptake of fluorescently labeled nanoparticles and the induction of costimulatory molecules (CD80, CD86) and cytokines (IL-1β and IL-12). We have previously demonstrated that positively charged PG nanoparticles induce increased expression of CD80 and CD86, robust secretion of IL-1β, and modest secretion of IL-12 [1]. The ability of the different nanoparticles to enhance the immune response will be tested using a model antigen (ovalbumin (OVA)) in a mouse model. BALB/c mice (n=8/group) will be injected intramuscularly with 10 µg of OVA and 200 µg of PG nanoparticles twice with a three week interval. Mice injected with OVA only or nanoparticles only will serve as negative control. The serum anti-OVA antibodies and T cell response to OVA will be determined two weeks after the second injection. Antibody titers will be converted into log10 values and analyzed by ANOVA followed by a Tukey's multiple comparison test. T cell-secreted cytokines (IFN-γ, IL-4, IL-17) will be determined by ELISA and the differences between the means will be determined by ANOVA followed by a Tukey's multiple comparison test.Objective 2. Twelve Yorkshire x Landrace pigs, each about 30 lbs, will be purchased from the Purdue Animal Sciences Research and Education Center. They will be tested for anti-influenza H1 antibodies prior to the purchase. Only pigs with negative titers will be used in the study. Pigs will be injected with PG nanoparticles only, influenza H1 protein, or nanoparticles mixed with H1 protein (n=4/group). The type of PG nanoparticles will determined based on the results from Objective 1. We have already demonstrated that positively charged nanoparticles have an adjuvant effect in mice and these will be the default option. The injection will be repeated after 3 weeks and blood samples collected after the first and second injection will be analyzed for anti-influenza H1 antibodies by ELISA and hemagglutination-inhibition (HI) test. The HI test will be conducted by Dr. Suresh Mittal.Objective 3. While positively charged PG nanoparticles can enhance the antibody response by themselves, it may be desirable to induce a stronger and tailored response by combining the nanoparticles with other immunostimulatory compounds such as Toll-like receptor (TLR) agonists. Because of the positive surface charge of the nanoparticles, negatively charged compounds such as nucleotides are particularly attractive candidates to include in such combination adjuvants. We will test poly-(I:C), a ligand for TLR3, and CpG oligonucleotides, ligands for TLR9. These ligands stimulate different intracellular signaling pathways and trigger different immunological mechanisms [3,4]. Combinations of nanoparticles and TLR ligands will be tested in vitro with dendritic cells as described under objective 1. Combinations that demonstrate synergistic interactions in vitro will be tested in vivo in mice using intramuscular and intranasal routes of immunization with OVA as antigen. Antibody and T cell responses will be analyzed as described under Objective 1.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Scientists in academia and industry working on vaccine discovery and development. Undergraduate, veterinary, and graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate and graduate students learn how to conduct immune assays including ELISAs, flow cytometry and Western blots, how to analyze RNA-seq data, and how to set up experiments to test vaccine efficacy in animals. How have the results been disseminated to communities of interest?Yes, through presentations at international (CRWAD) and regional meetings (Autumn Immunology Conference). What do you plan to do during the next reporting period to accomplish the goals?We expect to complete the bioinformatic analysis of the RNA-seq data of porcine dendritic cells stimulated with Nano-11 or Nano-11/poly(IC). We expect to start the intradermal and intranasal immunization experiments with a split virus influenza vaccine in pigs (delayed because of COVID-19).

Impacts
What was accomplished under these goals? We have demonstrated that the Nano-11 adjuvant activates swine monocyte-derived dendritic cells (moDCs) in vitro to secrete a variety of cytokines and by increasing the expression of CD80/CD86 and MHCII. We have activated moDCs with Nano-11 alone and with poly(I:C) and collected RNA for sequencing. Significant changes in mRNA expression were observed upon stimulation with Nano-11 and between Nano-11 and Nano-11/poly(I:C). The bioinformatics analysis is under way. We have further demonstrated that Nano-11 provides an effective delivery mechanism for cyclic dinucleotides. There is marked synergy between Nano-11 and these STING agonists.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: HogenEsch H, Gourapura R, Yao Y (2019) Improving vaccine performance with a novel phytoglycogen nanoparticle adjuvant. 100th annual Conference for Research Workers in Animal Diseases meeting. Chicago, November 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hernandez J, Mosley YC, HogenEsch H (2019) Intradermal delivery of a novel combination adjuvant promotes immunity without local adverse reactions. Autumn Immunology Conference, November 2019.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists in academia and industry working on vaccine discovery and development. Undergraduate, veterinary, and graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student is working on this project together with an undergraduate student. The students learn how to conduct immune assays including ELISAs, flow cytometry and Western blots, and how to set up experiments to test vaccine efficacy in animals. How have the results been disseminated to communities of interest?Through peer-reviewed publications and an oral presentation at the Modern Vaccines and Adjuvant Systems 2019 conference in September. What do you plan to do during the next reporting period to accomplish the goals?We will evaluate the effect of combining Nano-11 with the TLR3 agonist poly(I:C) on physical characteristics of the nanoparticles (size, zeta potential), and onthe activation of dendritic cells (costimulatory molecules, gene expression, cytokine secretion). We will also conduct further studies on intradermal vaccination of pigs with Nano-11 alone and in combination with other immunostimulatory molecules.

Impacts
What was accomplished under these goals? We have demonstrated that the Nano-11 adjuvant activates swine monocyte-derived dendritic cells in vitro to secrete a variety of cytokines. We have further demonstrated that Nano-11 can be used for intradermal vaccination in both mice and pigs, and enhances the immune response. This route of vaccination permits needle-free injection.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: 141. Dhakal S, Lu F, Ghimire S, Renu S, Laxmanappa YS, Hogshead BT, Ragland D, HogenEsch H, Renukaradhya GJ (2019) Corn-derived alpha-D-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization of pigs. Nanomedicine: Nanotechnology, Biology and Medicine, 16:226-235.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 142. Lu F, Mosley YC, Brown D, Carmichael B, HogenEsch H (2019) Formulation of aluminum hydroxide adjuvant with TLR agonists poly(I:C) and CpG enhances the magnitude and avidity of the humoral immune response. Vaccine, 37:1945-1953.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists in academia and industry working on vaccine discovery and development. Students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student is working on this project together with two undergraduate students. The two undergraduate students both graduated in the spring of 2018, and one of them started vet school in the fall of 2018. The students learn how to conduct immune assays including ELISAs, flow cytometry and Western blots, and how to set up experiments to test vaccine efficacy in animals. How have the results been disseminated to communities of interest?Through peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?We will investigate the efficacy of Nano-11 in combination with other immunostimulatory agents in mice and pigs using different routes of immunization.

Impacts
What was accomplished under these goals? We have demonstrated that an adjuvant composed of positively charged plant-derived nanoparticles, Nano-11, stimulates the immune response to protein antigens following intramuscular injection in pigs. In addition, Nano-11 shows promise as a mucosal adjuvant for intranasal immunization of pigs against influenza virus using a whole killed virus vaccine. The results from these studies are prepared for publication.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: 139. Dhakal S, Renu S, Ghimire S, Shaan Lakshmanappa Y, Hogshead BT, Feliciano-Ruiz N, Lu F, HogenEsch H, Krakowka S, Lee CW, Renukaradhya GJ (2018) Mucosal immunity and protective efficacy of intranasal inactivated influenza vaccine is improved by chitosan nanoparticle delivery in pigs. Front Immunol, 9:934.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: HogenEsch H, OHagan D, Fox C (2018) Optimizing the utilization of aluminum adjuvants in vaccines: You might just get what you want. Npj Vaccines 3:51