The role of b cell CUL4B in cross-protective… (F31AI194666) – Project Grant to University Of Florida

Abstract: Influenza a and b viruses infect the upper respiratory tract producing a highly transmissible infection. They infect millions of people and cause thousands of deaths in the united states annually. Seasonal vaccine campaigns are essential in preventing influenza infection and spread. Current vaccines work by eliciting robust antibody responses; however, they provide limited protection against emerging variants. Improved vaccines that could elicit cross-protection against new IAV variants would constitute a major advance, but little is known about how these antibodies develop. The overarching goal of this proposal is to elucidate b-cell intrinsic mechanisms underlying the production of cross-protective antibodies against influenza a virus (IAV). Infection or vaccination to IAV causes the secretion of antibodies by plasma cells. Prior to plasma cell differentiation, b cells endure multiple rounds of somatic hypermutation, proliferation, and selection (cyclic RE-entry) in germinal centers (GC) to produce high affinity antibodies. How GC b cells coordinate DNA damage and regulate cell cycle is not understood. Cullin 4B (CUL4B) is the backbone for an E3 ubiquitin ligase complex that ubiquitylates proteins for degradation by the proteosome. IT regulates DNA damage responses and cell cycle progression, but IT's role in b cells is unknown. The objective of this proposal is to determine the role of CUL4B in supporting GC b cell responses required for antibody-mediated cross-protection against IAV. In our preliminary studies, we showed that loss of b cell-intrinsic CUL4B causes a marked decrease in IAV-specific IGG titers, total and IAV-specific GC b cell populations, and increased morbidity. This supports the idea that CUL4B promotes antibody-mediated protection. In other studies, we showed CUL4B promotes formation of high affinity antibodies and regulates cell cycle. We hypothesize the ubiquitin ligase, CUL4B, promotes affinity maturation and cross- protective antibody production in GC b cells by regulating cell cycle in the face of DNA damage. In aim 1, we will define the role of CUL4B in germinal center b cell dynamics, the trajectory of GC b cells undergoing affinity maturation. GC b cells can DIE, RE-enter GCS (cyclic RE-entry), or differentiate and exit as memory or plasma cells. Using a brdu labeling approach and employing bulk-DNA sequencing of IAV-specific GCS, we will determine the effect of CUL4B on GC b cell cyclic RE-entry, differentiation, and expansion following IAV infection. We believe this will show CUL4B regulates cell cycle progression to promote cyclic RE-entry leading to affinity maturation. In aim 2, we will define the role of CUL4B in IAV cross protection. This occurs when a vaccine against one virus strain provides protection against a different strain. Using an immunization and challenge model of IAV, we will elucidate whether CUL4B is required for antibody-mediated cross protection. We believe CUL4B will promote cross protective antibody responses due to its reliance on extensive somatic mutations in GCS. Understanding how CUL4B mediated-ubiquitylation regulates affinity maturation in GC b cells will provide insight on mechanisms underlying cross-protective antibody production as a potential target for IAV vaccines.

Action	Obligated	Date
Amendment	$41,758	May 20, 2026
Amendment	$41,758	May 20, 2026
Original award	$0	May 20, 2026

The role of b cell CUL4B in cross-protective antibody responses to influenza a virus

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