Synthesizing virus-like particle conjugates for therapeutic and diagnostic purposes [electronic resource]

Virus-like particles (VLPs) are protein nanoparticles produced from capsid protein monomers of viruses. Although VLPs are structurally similar to viruses, VLPs do not contain the viral genome and are not infectious. As a nanoparticle, VLPs have unique properties, such as monodisperse size distribution, self assembly, stability, and repeated presentation of external and internal epitopes. Because VLPs are assembled from proteins, they can be produced using biological systems and their physical and chemical properties can be manipulated by altering the DNA sequence encoding the capsid monomer. These properties have made VLPs a subject of intense research in biological applications. The research in this thesis focuses on VLPs based on the MS2 virus and the Hepatits B (HepB) virus. The MS2 VLP forms from the self assembly of 180 capsid monomers, while the HepB VLP forms from the self assembly of 240 C-terminus truncated capsid monomers. The VLPs are produced using an E. coli based cell free protein synthesis (CFPS) system, which allows for the incorporation of non-natural amino acids into the protein. Specifically, an azide functional amino acid can be incorporated into a surface exposed loop of the VLP capsid monomer and we can use copper catalyzed azide-alkyne (3+2) cycloaddition ("click" reaction) to attach various ligands onto the surface of the VLP. Because of the diversity of potential ligands, the VLP can be tailored and fine-tuned for a variety of applications. One application of VLPs is the engineering and production of a VLP-based vaccine platform. Antigens specific to a disease, as well as immune stimulants, can be conjugated onto the surface of the VLP. The advantages of a VLP-based vaccine over traditional vaccines are that: 1) the size of the VLPs enables the VLPs to be channeled specifically to the lymph nodes when injected subcutaneously, 2) the repeated presentation of the antigens and immune stimulants on the same VLP increase the local concentration of these molecules when interacting with immune cells, and 3) the ability to tailor the type and concentration of immune stimulants to elicit a specific immune response for the treatment of a disease. These advantages over traditional vaccines allow for more protective immune responses against the antigen and for the possibility of eliciting immune response against antigens that are generally ignored by the immune system. We tested the efficacy of the VLP-based vaccines using the B cell lymphoma mouse model. B cell lymphoma occurs when a clonal population of white blood cells or lymphocytes becomes cancerous. One feature of B cell lymphoma is that the variable region of the surface antibody (known as the idiotype) is the same across all cancerous cells and unique from other non-cancerous B cells. This makes the idiotype an ideal target for the treatment of B cell lymphoma. Our treatment strategy is to conjugate the idiotype antibody variable region fragment (scFv) to the surface of a virus-like particle, as well as immune stimulants, such as granulocyte macrophage colony stimulating factor (GMCSF) and CpG DNA. In the first mouse trial, mice dosed with the VLP conjugated to the scFv, GMCSF, and CpG DNA had a humoral antibody response that was four times higher than mice dosed with the scFv alone. Also, the mice dosed with VLP conjugated to the scFv, GMCSF, and CpG DNA had significantly higher tumor challenge survival rates compared to the control mice. This study show that the VLP-based vaccine is feasible and has efficacy similar or better than traditional vaccination methods. After the first mouse trial, we made several improvements to the VLP vaccine platform. We produced a new HepB mutant (HepB SS1) that was stabilized with interdimer disulfide bonds. We improved the scFv conjugation to the new HepB SS1 mutant. We designed and produced flagellin for use as a vaccine adjuvant. With these improvements, we conducted a second mouse trial to test the efficacy of the VLP vaccine. In this mouse trial, mice dosed with VLP conjugated to the scFv and CpG DNA had similar humoral response and similar tumor challenge survival rate as the mice dosed with 38C13Id-KLH (positive control). We observed that flagellin acted as an immune inhibitor, rather than an adjuvant, when conjugated to the VLP vaccine. This study confirmed that the VLP-based vaccine can induce a strong and protective antibody response against lymphoma. For future work, it is possible to change the antigen and adapt the VLP-based vaccine for a variety of other diseases.

Keywords: Chemical Engineering.
Advisor: Swartz James R. primary advisor. Thesis advisor
Dunn Alexander Robert advisor. Thesis advisor
Smolke Christina D. advisor. Thesis advisor