BIOENGINEERING OF CHIMERIC ALPHAVIRUS VIRUS-LIKE PARTICLES AND THEIR APPLICATIONS

Abstract

From gene therapy to vaccines, the potential of alphaviruses as a versatile platform is just beginning to be explored. The findings in this thesis, adds newer insights ever-evolving field of diagnostics, antiviral drug discovery and hybrid vaccine design using bioengineered chimeric alphavirus virus-like particles. Alphaviruses are enveloped RNA viruses that infect both humans and animals and are spread by mosquitoes which leads to outbreaks of diseases such as Chikungunya, Eastern Equine Encephalitis, and Ross River Fever. Their clinical importance lies in the fact that they can cause a symptom ranging from mild fevers to severe encephalitis and arthritis. Therefore, understanding the clinical importance of alphaviruses is crucial for the development of effective diagnostic tests, antiviral drugs, and vaccines. Additionally, tracking and monitoring re-emergence of alphaviral infection is crucial for controlling their spread and minimizing the impact they have on public health. Although in recent years, alphaviruses have been used as a platform for the production of viral vectors and enveloped virus-like particles (VLPs). From gene therapy to vaccines, alphaviruses offer endless possibilities for improving human health and advancing scientific discovery, through gene therapy, drug delivery, cancer therapy and vaccine designs. Virus-like particles (VLPs) are structures that mimic the external appearance of viruses lacks completely viral genetic material. VLPs have numerous applications in various fields, such as vaccine development, drug delivery, and biotechnology. One of the most promising uses of VLPs is in vaccine development. Because VLPs closely resemble the actual virus, they can elicit an immune response that is similar to that generated by a live virus vaccine. This allows for the development of safe and effective vaccines that do not pose the risk of spreading the disease. Another important use of VLPs is in drug delivery. VLPs can be engineered to carry drugs, enzymes, or other therapeutic agents, allowing for targeted delivery to specific cells or tissues. This increases the efficacy of the drug and reduces side effects. Chimeric alphavirus-based VLPs are structures developed by recombining proteins from different alphaviruses to generate a new hybrid structure. These unique and versatile chimeric VLPs have a wide range of applications in fields such as vaccine development, drug delivery, and basic research. This thesis, will focus on chimeric alphavirus-based VLPs in field of diagnostics, antiviral drug discovery and hybrid vaccine design. Fluorescent (mCherry protein), luminescent (NanoLuc) and immunogenic antigen RBD of SARS-CoV-2 has been utilized to design and generate unique and versatile chimeric chikungunya virus-like particles. It was rather encouraging to observe promising results in successfully generating fluorescent, luminescent and immunogenic alphavirus-based virus-like particles by exploiting various techniques like live-cell fluorescent imaging, cryoEM, surface plasmon resonance (SPR) etc. CryoEM micrographs of mCherry CHIK-VLPs and NLuc CHIK-VLPs purified from transiently transfected 293Ts, reveals spikey particles of 60 nm and 50~70nm respectively. As our understanding of VLPs and their potential applications continues to grow, it is likely that we will see more innovative uses for these fascinating structures in the future. The chimeric alphavirus virus-like particles technology platform unlocks the potential of virus-like particles as versatile nanomachines which opens up new doors in vaccine development, targeted drug delivery and diagnostic tools. Chapter 1: Reviews the literature related to VLPs, alphaviruses and the significance of structural proteins of CHIKV in VLPs production. The chapter briefs about advancement in virus-like particles technology and its application in various domains of bio-medical science. This chapter discusses in detail, the types of VLPs and various VLP-based vaccines that are in clinical trial and those which have already been approved for use in market. In this chapter, the significance and need for chimeric alphavirus VLPs will also be explained. This chapter discusses, why use of reporter-tagged VLPs is better than reporter-tagged viruses. Chapter 2: Reports the production, purification and characterization of chimeric reporter-tagged chikungunya virus-like particles. This chapter describes in detail, the optimized workflow for expression, production, purification and characterization of purified NLuc-tagged CHIK-VLPs and mCherry-tagged CHIK-VLPs. Western Blot, IFA and live-cell imaging was performed to confirm the expression of reporter-tagged CHIK-VLPs in transfected HEK293Ts. Cryo-electron microscopy (CryoEM) has been performed to reveal particle diameter as ~67 nm and 56 to 70 nm, respectively, for NLuc CHIK-VLPs and mCherry CHIK-VLPs. Chapter 3: This chapter demonstrates the applications of chimeric reporter-tagged CHIK-VLPs in clinical diagnostics and viral entry-inhibitor screening. mCherry (26.7 kDa, fluorescent protein) and NanoLuc (NLuc, 19.1 kDa, monomeric luciferase enzyme) has been used as reporter proteins tags for generating mCherry CHIK-VLPs and NLuc CHIK-VLPs. Using reporter-tagged CHIK-VLPs, we have established a faster and more sensitive detection method for screening alphavirus entry inhibitors. NLuc-CHIK VLPs have been exploited for detection and titration assay for CHIKV-antigen and anti-CHIKV antibody in clinical samples. In this chapter, detailed methodology and results for CHIKV-antigen and anti-CHIKV antibody detection and titration assay is described. Similarly, the alphavirus entry-inhibitor screening optimized assay protocol and results has been discussed in detail. Chapter 4: Reports the production, purification and characterization of chimeric alphavirus-coronavirus virus-like particles (ChAC-VLPs). This chapter describes in detail the optimized workflow for production, purification and characterization of purified ChAC-VLPs. The detailed methodology describes mammalian (HEK293T) expression system for production and purification of ChAC-VLPs. The structural integrity and particle homogeneity of ChAC-VLPs was studied using cryo-electron microscopy and DLS. To further validate the co-expression of CHIKV structural proteins and RBD of SARS-CoV-2 in purified ChAC-VLPs; western blot, SDS-PAGE and Indirect IFA (immunofluorescence assay) have been performed. Furthermore, various in vitro assay validates the multimeric epitope availability of RBD-E2 fusion protein displayed on the surface of ChAC-VLPs. Chapter 5: This chapter describes, mice immunization experiments for ChAC-VLPs for its potential application in hybrid vaccine design for CHIKV and SARS-CoV-2. The immunogenicity assessment of ChAC-VLPs have been performed on swiss-albino mice. This chapter, explains the workflow for ELISA, surface plasmon resonance (SPR) and plaque reduction neutralization (PRNT) experiments for assessment of anti-RBD and anti-E2 antibody generated in immunized mice sera. RBD and E2 surface glycoprotein are the immunodominant antigen of SARS-CoV-2 and CHIKV respectively. ChAC-VLP immunized mice were assessed for antibody against both the antigens. Potent neutralizing activity is observed for CHIKV through PRNT assay; SPR and ELISA determined anti-RBD antibody in ChAC-VLP immunized mice sera. Hence ChAC-VLPs can be a potential hybrid vaccine candidate for SARS-CoV-2 and CHIKV infections. This chapter discusses the future directions in hybrid vaccine development using ChAC-VLPs. Chapter 6: Concludes the studies performed in the thesis. Reporter-tagged CHIK-VLPs and immunogenic ChAC-VLPs have been successfully designed, produced and purified. Reporter-tagged CHIK-VLPs have been applied for rapid CHIKV-diagnostics and viral entry inhibitor assay. Immunization of mice with ChAC-VLPs have shown potent CHIKV neutralizing and antibody were raised against RBD of SARS-CoV-2. Through CHIK-VLP based platform, medical diagnostics, antiviral drug discovery and hybrid vaccine candidate has been unravelled. This chapter discusses the future prospects of chimeric CHIK-VLPs in medical biotechnology field.