Degree Type

Dissertation

Date of Award

2015

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Genetics

First Advisor

Michael W. Cho

Abstract

As one of the world’s most devastating viruses, HIV-1 has killed more than 39 million people, and around two million cases of newly infected individuals are recorded every year. However, no effective vaccine has been developed to stop this pandemic since its onset in the 1980s. Since vaccine development is moving slowly, delivery platforms as an essential element to enhance both the efficiency and efficacy of vaccines have drawn increased attention. Gold nanoparticles (GNPs) as a novel delivery platform has been studied in drug and vaccine delivery. My research goal is to apply this delivery platform in the AIDS vaccine development field for HIV-1 envelope protein-based subunit antigens.

We studied different conjugation methods to load antigens on GNPs. First, we tried to directly link an antigen onto GNPs through an introduced cysteine mutation in the antigen, since the thiol group of cysteine can form a covalent bond with GNP. In this direct-linking study, we made several antigen variants with cysteine introduced in different positions of the original antigen. All cysteine variants can be successfully loaded on GNPs, with neutralizing epitopes exposed after bond on GNPs. We found that the terminal cysteine variants (with the cysteine mutation on either end of the antigen) can elicit high antibody response in rabbits; however, the antibody responses from the internal cysteine variants (with the cysteine mutation inside of the antigen) are exceptionally low. We suspected that the antigens loaded on GNPs trough the internal linkage might be too close to the GNP surface, therefore, it might be difficult to cleave these protein antigens from the GNPs in vivo by the protease in the endosome, which could result a low immune response.

To further enhance the antibody response, we hypothesized that a spacer between the GNP and antigen might be needed instead of direct linkage. We speculated that a linker between the antigen and GNP might promote antigen processing in the immune system after the antigen-loaded GNPs have been taken by the antigen presenting cells (APCs), which could assist in eliciting high immune responses. Therefore, we examined two linkers for our antigens to attach onto the GNPs. Again, our antigen can be successfully loaded on the GNPs through these linkers, with the critical neutralizing epitope exposed even after loading on the GNPs. After the pilot animal experiment, we chose the N-[Nα,Nα-Bis(carboxymethyl)-L-lysine]-12-mercaptododecanamide (SH-NTA) linker to continue our immunization studies in both mice and rabbits. As expected, compared to the protein-only immunized group, the protein-on-GNP group exhibited a much higher antibody titer reaching 107 after only three immunizations in two animal models. Neutralizing activity was also detected in rabbits. Our result suggests that the GNP-NTA system as a delivery platform could preserve the critical epitopes of the loaded antigen as well as elevate their antibody responses. Since our GNP-NTA platform was designed to deliver any protein-based antigen, the positive results from our study indicate that our GNP-NTA delivery system has great potential to be applied in protein-based vaccine development.

Copyright Owner

Feng Lin

Language

en

File Format

application/pdf

File Size

118 pages

Share

COinS