Pseudotyped lentiviral systems for studying viral entry proteins from emerging viruses with pandemic potential

Abstract

Viral entry proteins facilitate viral entry into host cells through receptor binding and membrane fusion. They play a critical role in the viral life cycle and, as prominent surface proteins, are common targets of the host immune response. Mutations in viral entry proteins can allow viruses to infect new hosts (including humans), better spread between hosts, or evade immune responses and some therapeutics. A better understanding of how viral entry proteins interact with host proteins, elicit host antibody responses, or are affected by mutations is important for being tackling current and emerging viral threats. However, viral entry proteins are also heavily glycosylated, multimeric, metastable proteins that are challenging to work with. Furthermore, studying viral entry proteins in their native viral context is challenging due to inefficient reverse genetics systems or the need to work under high biosafety level containment. As such, many viral entry proteins—especially those from emerging viruses—cannot be studied with many high- throughput experimental techniques, such as mutational scanning experiments. Pseudotyping viral entry proteins on easier- and safer-to-work-with viral particles is one way to facilitate research of viral entry proteins from emerging viruses. When SARS- CoV-2 was first identified in late 2019, there was an urgent need to develop tools to safely and easily study the virus. Working with SARS-CoV-2 itself requires a biosafety level 3 facility, but pseudotyping its entry protein, spike, onto lentiviral particles allows SARS-CoV-2 spike to be studied under commonly available biosafety level 2 conditions. In the following chapters, I describe a protocol for pseudotyping lentiviral particles with SARS-CoV-2 spike to facilitate research into this emerging virus. I also describe the use of spike-pseudotyped lentiviral particles to investigate the neutralizing antibody response to SARS-CoV-2 and the effects of mutations to spike on its function as a viral entry protein. Spike-pseudotyped lentiviral particles are especially useful for measuring SARS-CoV-2-neutralizing antibodies and I provide a detailed protocol for a SARS-CoV-2 neutralization assay using spike-pseudotyped lentiviral particles I then use this assay to investigate the dynamics of the neutralizing antibody response to SARS-CoV-2 in the first several months following infection. Titers of SARS-CoV-2 neutralizing antibodies decline modestly from ≈1 to ≈3 months post symptom onset, which is typical of the neutralizing antibody response to other acute respiratory viruses. The SARS-CoV-2 pandemic has also provided an example of the importance of prospectively characterizing the effects of mutations to viral entry proteins. Pseudotyped lentiviral particles provide a genetically tractable system for assessing the effects of mutations to viral entry proteins from emerging viruses at high-throughput. As such, I have worked to develop a pseudotyped lentivirus-based system for screening the functional and antigenic effects of mutations to viral entry proteins from emerging viruses in high-throughput. This system is still being developed, but I briefly discuss current progress and describe a Python package I helped write to facilitate the analysis of such deep mutational scanning experiments. In summary, the following chapters describe the use of pseudotyped lentiviral particles as a a flexible, rapidly deployable tool for studying viral entry proteins from emerging viruses, such as SARS-CoV-2. Using spike-pseudotyped lentiviral particles, I contributed to some of our earliest understanding of the dynamics of the neutralizing antibody response to SARS-CoV-2 in the first several months following infection. Nonetheless, I think the full potential of using pseudotyped lentiviral particles to study viral entry proteins from emerging viruses has yet to be realized. As such, I also discuss current progress (including computational tools) and future work towards using pseudotyped lentiviral particles to measure the functional and antigenic effects of mutations to viral entry proteins from emerging viruses at high-throughput.