Production of pseudoviral particles to evaluate neutralizing antibodies

• Grant number: 23/11334-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2023
• End date: August 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Geraldo Santana Magalhães
• Grantee: Thays Duarte de Oliveira
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 21/11944-6 - Continuous improvement of vaccines: Center for Viral Surveillance and Serological Assessment (CeVIVAS), AP.CCD

Abstract

Serological assays are essential for tracking the epidemiological spread of a virus and evaluating mass vaccination programs by quantifying neutralizing antibody responses against antigens on the viral surface. However, although there are now well-characterized, high-throughput methods (such as ELISA assays) to measure total antibody binding to viral protein epitopes, quantifying neutralizing antibody activity is more difficult. The most biologically relevant method is to directly measure how antibodies or sera inhibit infection of cells by the replication competent virus. These live virus assays are now being performed to quantify neutralizing activity in serum from infected patients or to characterize the potency of individual antibodies. These wild virus assays are now being performed to quantify neutralizing activity in sera from infected patients or to characterize the potency of individual antibodies. However, the throughput and affordability of live virus neutralization assays are limited by the fact that the virus is a high-level biosafety agent that must be handled in specialized facilities. An alternative approach that alleviates these biosafety limitations takes advantage of the fact that neutralizing antibodies target glycoproteins on the surface of the virus. These glycoproteins project prominently onto the surface of virions and are involved with binding to the target cell. In this way, the SARS-CoV-2 Spike glycoprotein or Influenza hemagglutinin A (HA) can be "pseudotyped" into safer non-replicating viral particles, replacing the endogenous entry proteins of these viruses, thus making the infection of these particles in the Spike- or HA-dependent cells. In this regard, HA genes have been used to make HA-pseudoviruses to facilitate assessments of how different particular mutations affect neutralization. Similarly, the spike (S) glycoprotein of coronaviruses such as SARS-CoV-2, which facilitates viral entry into host cells, has been used to form pseudoviruses. Due to the potential of pseudoviruses to mimic the entry mechanisms of the wild virus and to circumvent the high biosafety requirements, this project aims to construct these pseudoviral particles containing the variant glycoproteins of SARS-CoV-2 and Influenza A, from strains detected by genomic surveillance and utilize these pseudoviruses to study neutralizing antibody responses in a biosafety level 2 laboratory. data on the development of immunity in viral epidemics and potentially screening donors for passive transfer of convalescent plasma. (AU)