Highly Multiplexed Detection of Immune Responses to Emerging Infectious Diseases via Lentiviral Surface Display

Background: The ongoing global SARS-CoV-2 pandemic has made clear that new approaches are needed to study immune responses to infectious diseases. Analyzing antibody responses in serum is perhaps the most tractable possible measurement, due to its long duration, stability for collecting and storing samples, and importance in predicting protection from disease. While current approaches to monitor antibody reactivity such as ELISA can sensitively detect immune responses, they are not inherently able to be multiplexed and are not suited for examining immune effector functions. A 2017 Discovery Grant awarded to our group led to the development of a lentivirus targeting strategy that enables the specific infection and identification of T cells through repurposing their antigen receptor as a viral entry receptor. This work led to our development of a novel, sensitive, and versatile viral pseudotyping strategy. In this application, we present preliminary evidence that such an approach can readily be adapted for serology, providing the ability to quantitatively examine hundreds serum presence, neutralization, and effector functions against hundreds of viral variants simultaneously. Relevance to Topic Area(s): Our proposal directly addresses the topic of Emerging Viral Diseases. The ability to rapidly assess the presence, scope, and magnitude of antiviral serological responses is key in managing a pandemic. Our proposed work can enable such measurements with a breadth and depth not feasible by current means. Our work can also directly measure correlates between antibody recognition, effector functions, and disease outcome, addressing a direct area of focus. Hypothesis/Objective(s): This proposal aims to create a novel method, termed ARCADE (Antigen Reactivity Characterization by Antibody-Dependent Entry), capable of conducting multiplexed serology at scale. We will validate and benchmark an approach capable of quantitatively tracking the abundance, neutralization ability, and effector functions of antibodies in serum for hundreds of distinct pathogens at once. Specific Aims: (1) To develop, benchmark, and implement Hybrid Pseudotyping strategy for Serology at Scale. (2) To apply coronavirus variant libraries for multiplex surrogate neutralization assays (SNA) for functional assessment and predictions of viral escape. (3) To extend viral libraries to sort and sequence B cells for the discovery of novel antibody sequences. Study Design: We will design and construct a library of "hybrid pseudotyped" lentiviruses that are able to sensitively detect either antibody binding, binding to a cellular entry receptor, or disruption of cell entry via next-generation sequencing. This approach will initially focus upon currently described SARS-CoV-2 variants of concern. We will optimize protocols to maximize sensitivity and selectivity for detection of antibodies able to bind SARS-CoV-2 S protein, and for detection of neutralization ability. The same approach will be adapted to examine resistance to current and putative escape mutations, to decouple antibody-based effector functions, and to identify the sequences of cells producing desirable antibodies. Once validated, we will use our approach to examine multiple patient cohorts in an attempt identify novel correlates of immune protection or disease severity. Impact: Our proposal will enable rapid, quantitative, and low-cost analyses for serum reactivity. We anticipate that it would enable the detection of serum reactive to hundreds of full-length pathogen proteins, multiplexed to the study of hundreds of patient samples in a single experiment, all using common reagents and $10-$20/sample of next-generation sequencing. The method can also be extended to newly emerging pathogens in a matter of weeks. Such analyses can greatly aid in assessing previous exposure to pathogens, and designing and assessing the performance of vaccines or other drug candidates. Relevance to Military Health: U.S. Service Members are regularly placed into scenarios where they could encounter unknown pathogens. Understanding the nature of an effective immune response could be greatly beneficial to safeguarding that individual as well as for creating prophylactic treatments for pathogens going forward. Less