Development of protein-based therapeutics and vaccines
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Pendyala, Geetanjali
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Abstract
Engineered proteins play an important role in the development of therapeutics and subunit vaccines. The objective of this thesis is to design and develop protein-based vaccines and therapeutics using techniques such as site-specific modification, click chemistry, bioconjugation, and the multivalent display of antigens on virus-like particles (VLPs). In Chapter 2, we developed a VLP-based vaccine for malaria by presenting multiple copies of a chimeric circumsporozoite protein (CSP) antigen on SpyCatcher-mi3 nanoparticles. CSP is a surface protein expressed during the sporozoite stage in the life cycle of Plasmodium falciparum (Pf) malaria that causes considerable mortality worldwide. The chimeric PfCSP (cPfCSP) antigen incorporates the important “T1/junctional” epitope that is the target of potent neutralizing antibodies. Our vaccine candidate demonstrated high and durable IgG antibody levels and a balanced antibody response against the T1/junctional region as well as the (NANP)n repeats in mice. Moreover, the antibody concentration elicited by immunization was significantly greater than the reported protective threshold defined in a murine challenge model. In Chapter 3, we designed Staphylococcus aureus vaccines with cross-reactivity against multiple toxins that S. aureus generates to evade the host immune system. Three cytotoxin components (LukD, LukF, HlgB) share ~80% amino acid sequence identity. Though α-Hemolysin (Hla) cytotoxin is largely different from LukD, LukF and HlgB (~25% shared amino acid sequence identity), they contain a common conformational epitope that binds to neutralizing antibodies. Leveraging this similarity, we displayed multiple copies of LukD or Hla on SpyCatcher-mi3 nanoparticles and compared the breadth of the antibody response elicited by different vaccination schemes. In Chapter 4, we presented a strategy to incorporate non-canonical amino acids into lysostaphin, an enzyme targeting the cell wall of Staphylococcus aureus, while retaining its stapholytic activity. We used this approach to successfully generate active variants of lysostaphin that incorporate para-azidophenylalanine. Addition of this “reactive handle” facilitated the orthogonal site-specific modification of lysostaphin variants with polyethylene glycol (PEG) using copper-free click cycloaddition. Our results showed that PEGylated lysostaphin variants could retain their stapholytic activity. However, the extent of retention depended on the location of the modification site and the molecular weight of PEG. In Chapter 5, we designed VLP-based coronavirus vaccines. Multiple copies of the Receptor binding domain (RBD), Spike (S) protein and S2 domain of SARS-CoV-2 were conjugated to SpyCatcher-mi3, and mice were immunized with the constructs intranasally. Systemic immune response was characterized, and the mice were challenged against SARS-CoV-2 to evaluate protection.
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2023-12-07
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Dissertation