Engineering Antigen Display for a PRINT Particulate Dengue Subunit Vaccine Platform

摘要

Traditional vaccines consist of live attenuated pathogens that will always require a balance of attenuation, to achieve an adequate safety profile, and immunogenicity, to achieve an adequate immune response. Additionally, live vaccines are not viable for pathogens that are too dangerous or evade our immune system. Subunit vaccines consist of purified soluble antigens that can be produced using recombinant methods, providing both safety and manufacturing advantages over traditional vaccines. However, subunit vaccines often lack immunogenicity and therefore efficacy. Particulate platforms have revolutionized drug delivery and improved the efficacy of subunit vaccines by transporting antigens to their target locations or immune cells. The immunogenicity of nanoparticle platforms is being enhanced further by formulation developed for the co-delivery of adjuvants. While, antigen delivery and immunogenicity are being boosted, little attention is being paid to the presentation of antigens. Surface receptor interactions are critical to the response of immune cells and highly neutralizing structural epitopes are being found in many modern vaccine targets (HIV, influenza, dengue, zika, etc.). The surface conjugation of antigens to particulate platforms provides the opportunity to control and optimize their display to produce the maximum immune response.;The overall goal of this work is to test the effects of varying antigen display characteristics for surface conjugated particulate platforms and develop a surface conjugated particulate platform displaying a recombinant dengue ectodomain (E) antigen. Reproducible polymeric hydrogel particles of exact size, shape, and composition produced using Particle Replication in Non-wetting Templates (PRINT) technology that have previously been shown to drain to lymph node and induce a response to surface conjugated model antigens will be used. The effects of antigen density and the proximity of antigen conjugation to the particle surface were evaluated in vitro and in vivo. Increased antigen density and linker length on the particle resulted in improved cellular responses in vitro and higher influenza neutralizing antibody titers towards hemagglutinin (HA) antigen in vivo. With a platform demonstrated to elicit a stronger response with improved antigen display, we sought to develop a surface conjugated dengue E subunit particulate platform. The antibody response to dengue E depends strongly on its presentation and structure. Strongly neutralizing antibodies towards epitopes formed by quaternary dengue E structures are rapidly becoming targets of vaccines. Initial efforts, using previously effective methods to generate dengue E particles, produced a non-dengue specific and non-neutralizing antibody response. By optimizing the conjugation buffer and developing methods of examining the structure of dengue E conjugated to the particle surface new particulate formulations were produced that induced dengue-specific, and neutralizing, antibody titers. Using the developed platform, a conjugation method for the site-specific conjugation of dengue E at its c-terminus allowed for the assembly of dengue E dimers and display of a highly neutralizing quaternary epitope the particle surface. This work demonstrates the importance of antigen display for particulate platforms and develops a surface conjugated PRINT particulate platform that allows for the display of highly neutralizing dengue E quaternary structural epitopes.