Development of HIV-1 and SARS-CoV-2 Pseudo-Typed Viral Particle Systems for Studying Virus Infectivity

Abstract

The ongoing global SARS-CoV-2 pandemic highlights the importance of developing innovative technologies in monitoring viral outbreaks. One such platform is the generation of Pseudo-typed Virus Particle (PVP) systems, which provides a safe alternative to utilising replicative competent viruses in studying virus phenotypes. PVPs provide an immunogenic, high-throughput, and resilient system that can result in the rapid testing of emerging viral phenotypes. This thesis encompasses the development of HIV-1-based PVP systems as a platform for pseudo-type engineering and generating improved tools for studying HIV-1 and SARS-CoV-2 infectivity. Initially, a THP-1 cell model system was developed to study HIV-1 infection of physiologically relevant macrophage (MQ) and dendritic cells (DCs). The results identified that PVP generated with the pSG3 vector backbone were more infectious than those generated utilising the pNL4-3-Luc system. This highlights that PVP can be used to successfully study HIV-1 infection of THP1-MQ and THP1-DCs. Second, a cloning strategy was developed with the aim of generating PVP with improved monitoring capacity by generating new plasmids expressing the luciferase reporter protein in different insertion sites on the backbone plasmid. One generated plasmid (pSG3-Luc-4) showed a high level of luciferase protein expression following infection. It was also shown that when generating PVP in conjunction with HIV-1 Tat expressing plasmid, higher levels of PVP production and infectivity could be generated, allowing for higher yields of PVP stocks. Third, a PVP assay using a SARS-CoV-2-S envelope (Env) protein was developed and tested for infectivity in various cell-types. These were found to be a powerful tool for assessing neutralising antibody responses raised against SARS-CoV-2 as well as detecting viral entry at the single-particle level using fluorescent SARS-CoV-2 PVPs. The results demonstrated that SARS-CoV-2 PVPs can be used for studying receptor usage, mechanisms of infection, and testing of neutralising antibodies, which is vital when assessing immune responses mounted against new vaccine candidates. Finally, the efficiency of SARS-CoV-2 PVP production was compared when generated in different producer cell-types and how it affected the infection characteristics of the resultant virus. High levels of viral p24 antigen were found in PVPs produced using 293T and 293T-ACE2 cells. It was observed that the expression of ACE2 in the producer cells can influence the phenotype of PVPs generated. Surprisingly, it was revealed that the virus generated in cells expressing the ACE2 protein produced infectious PVP in the absence of viral Env expression. These results suggest that ACE2 incorporation within PVPs may lead to the infection of numerous cell-types lacking ACE2 expression and may potentially explain for the expansion of cell tropism observed in SARS-CoV-2 infection. Overall, the results presented in this thesis describe the expansion and improvement of PVP systems in studying HIV-1 and SARS-CoV-2 viral phenotypes. These findings underscore the potential of utilising PVPs in studying viral outbreaks and their unique benefits when aiming to better understand viral entry, cell-type of infection and study of host viral interactions. Such research is crucial in the ongoing fight against the current global SARS-CoV-2 pandemic and in preparing for future viral outbreaks.