This Small Business Innovation Research Phase I project proposes to demonstrate the feasibility of using nanoparticles as an adjuvant for enhanced antibody production. A number of adjuvants are commercially available for use in animals to boost antibody production, but most of them pose potential hazards such as excessive inflammation leading to the formation of chronic granulomas, sterile abscesses, and/or ulcerating tissue necrosis. Most importantly, low antibody titer causes lower yield of antibody, which increases the need to use more animals to produce antibodies. Thus, in Phase I of this project, we will study the effects of cadmium-free nanoparticle structure, surface coating, size, surface charge, and antigen density on animal immunogenicity. We will optimize the formulation of the nanoadjuvant (covalent or charge-charge nanoparticle-antigen interaction) and study the bio-affinity of the antibodies produced. We expect that the proposed nanoadjuvant will increase the immunogenicity of the antigen and that the antibody titer will be 10 times higher than that without the nanoadjuvant.
The broader impact/commercial potential of this project relies on antibody products that are the fastest growing, most active, and most promising class of pharmaceuticals, especially with the growth of human immunotherapy. The worldwide revenues of antibody-based therapeutic treatments generated around $20 billion in 2007 and were growing at 14% a year. Antibodies belong to a safe and target-specific category of pharmaceuticals that have a relatively high success rate from early clinical studies to licensure. The proposed inorganic nanoparticle-based adjuvant can dramatically increase the immunogenicity of antigens and produce high antibody titers. Successful development of this project will greatly reduce the cost of antibody production to benefit human healthcare and biomedical research. Furthermore, it can eliminate pain, inflammation and other side effects to animal, as well as reduce the number of animals used.
1. Project outcomes or findings that address the intellectual merit and broader impacts of the work as defined in the NSF merit review criteria. This project is focused on the ability of inorganic nanoparticles to help induce antibody production in animals. The nanoparticles that were tested were water soluble inorganic nanoparticles including quantum dots made of cadmium selenide/zinc sulfide and copper indium sulfide, magnetic iron oxide, gold, and silver nanoparticles. The NPs were tested in animals such as rabbit, mouse, chicken, and Aotus monkeys. The antigens that were tested wee proteins included mouse IgG, bovine serum albumin, human IgG, chicken ovalbumin, and recombinant merozoite surface protein of Plasmodium falcipatum, a protozoa that causes malaria. The NPs were first synthesized in organic solvent before these were converted into water soluble forms using a polymer coating that introduced hydrophilicity on the NPs surface. The water soluble NPs that contained carboxyl groups on the surface were characterized and tested for stability before use. After establishing the size/shape, hydrodynamic size, zeta potential, conjugation ability, and stability in physiological buffers, the NPs were tested human cell lines to establish the levels that were toxic. Non-toxic levels were used in the animal studies. The NPs were attached to the antigens to form the nanoparticle-antigen formulation. The nanoparticle-antigen formulation was prepared using covalent attachment with ethyldimethylcarbodiimide hydrochloride. The animals (rabbits, chicken, mouse, Aotus monkeys) did not show adverse reaction to all the NPs studied in this project even after an extended period of observation beyond 6 months. Serum of animals plus the eggs of chicken were collected and evaluated for antibody production. Antibody titers were evaluated using standard 96-well plate enzyme linked immunosorbent assay protocols. The antibody titers using the nanoparticle-antigen formulations indicated that the NPs functioned as adjuvants for the antigens causing an increased antibody production compared with the antigen alone. Activity of the antibodies was evaluated using enzyme linked immunosorbent assay, fluorescence immunoassay, and parasite growth inhibition. The antibodies produced using the nanoparticle-antigen formulations were very active. In addition, the antibody produced against rMSP showed very potent inhibitory effects on the P. falciparum parasites. Toxicity Studies showed no abnormalities in NP immunized animals. Organs of animals treated with NPs showed no damage and/or abnormalities in comparison with the control as shown in Figure 1. This is possibly due to the very low dose at which the NPs were used in the nanoparticle-antigen formulations. The results of these studies in a broader perspective showed that: 1) The nanoparticles studied were non-immunogenic on the animals studied. 2) The antigens attached to the nanoparticles did not produce adverse effect to the animals and did not cause any organ damage. 3) The antigens attached to the water soluble nanoparticles elicited higher (as much as 2 orders of magnitude) antibody titers than commercially available adjuvants that are in oil and water emulsion causing pain, inflammation and granuloma formation at the site of injection. 4) The antibodies produced in the presence of the nanoparticles were actively immunogenic against their corresponding antigen. 5) The antibodies produced were easily conjugated with common reporter molecules such as enzymes, organic dye, quantum dots, and iron oxide nanoparticles. 6) The conjugated antibodies were active when used in a 96-well enzyme linked immunosorbent or fluorescence assay. 7) The conjugated antibodies were active as primary and as secondary antibodies. 8) The conjugated antibodies were useful for imaging applications. 9) The antibodies against the model vaccine candidate were active in inhibiting the disease causing organism. 10) The antibodies were active in sequestering cancer cells, respectively. 11) The nanoparticle~antigen formulations can be used to deliver antigens in animals and produce enhanced immune response that can be harnessed for antibody production or for vaccine/drug delivery. 2. Publications and patent Published: Pusic, K., Xu, H., Stridiron, A., Aguilar, Z., Wang, A., Hui, G., Blood stage merozoite surface protein conjugated nanoparticles induce potent parasite inhibitory antibodies, Vaccine, 2011, 29:8898-8908. Submitted: Pusic, K., Aguilar, Z., Kobuch, S., Xu, H., Tsang, M., Wang, A., Hui, G., Iron Oxide Nanoparticles as a Clinically Acceptable Delivery Platform for a Recombinant Protein Blood-Stage Malaria Vaccine, Submitted to ACS Nano, Jan. 2012. Z. Aguilar, A. Wang, G. Hui, H. Xu, K. M. Pusic, Nanoparticle based Immunological Stimulation, US Patent Application #61434073, January 16, 2012
