(1001-B) A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Neutralizing antibodies block the virus from infecting its target cells. Measuring neutralizing activity in highly reliable and reproducible assays that are amenable to standardization and automation is key. A typical workflow of in vitro assay to determine the half-maximal inhibitory concentration (IC50) of a neutralizing antibody involves multiple labor-intensive steps: 1) preparation of an antibody dilution series, which are incubated with live virus; 2) transfer of antibody-virus mixtures to cultured cells; 3) detection of virus-infected cells (e.g., cytopathogenic effects or probing a viral protein) at 1-4 days post-infection; and 4) infer the IC50 from dose-response curve fitting all the measurements. However, due to the use of live virus, it requires increased biosafety containment and is difficult to standardize and/or automate. Influenza virus is an orthomyxovirus with a segmented, negative-sense, single-stranded RNA genome, which allows the virus to reassort segments with each other and become extremely diverse. Seasonal influenza viruses (e.g., H3N2 and H1N1 influenza A, or influenza B) evolve rapidly in humans causing annual seasonal epidemics, whereas other viruses can emerge from the animal reservoirs and cause pandemics in humans. Next-generation vaccines should be able to elicit antibodies able to neutralize not only seasonal viruses but also viruses circulating at the human-animal interface, such as highly pathogenic avian influenza H5Nx and H7N9, or viruses which were able to infect humans in the past and still circulate in the animal reservoirs (i.e., H2N2). To facilitate next-generation influenza vaccine effort, we developed a safe and high-throughput neutralization assay, which can be used in laboratories with standard biosafety containment. Using influenza reverse genetics, we rescue replication-restricted reporter (R3) influenza viruses in which one viral gene essential for virus replication (e.g., viral RNA polymerase PB1) is replaced with a fluorescent reporter gene, and propagate R3 viruses in cells expressing the deleted gene in trans. R3 viruses are safe as they cannot replicate in cells lacking the deleted viral gene, and the R3 virus infected cells can readily detected by live fluorescence imaging. Using this approach, we prepared a panel of more than 60 R3 viruses that include seasonal influenza A and B viruses representing their evolution in humans and animal influenza viruses of concern. We used the panel of R3 viruses in high-throughput neutralization assay using 384-well plates for in-depth profiling of monoclonal antibodies isolated from vaccinees of next-generation influenza vaccine candidates developed by the Vaccine Research Center. To increase throughput, we are currently working to setup the assay using 1536-well format. Our assay system offers deeper understanding of the antibody-virus interactions and facilitates in-depth characterization of infection- and vaccine-elicited neutralizing antibody response, which in turn will provide insights towards developing the next-generation influenza vaccines.