Wobble Vaccines: Eliminating Immunodominance and Promoting Cross-Strain Protection Through Flexible mRNA Vaccine Library Encoding

The COVID-19 pandemic has brought the need for innovative vaccination platforms capable of rapidly curbing emerging viral diseases. A bright spot has been the emergence of mRNA vaccination platforms as robust, effective, flexible, and safe. However, while these platforms show incredible promise, viral variants have already resulted in reduced vaccine efficacy. This highlights an ongoing challenge in next-generation vaccine design - epitope targeting. Analyzing SARS-CoV-2 evolution from 2019, particularly variants identified by the Centers for Disease Control and Prevention (CDC) as "variants of concern," reveals specific loci within the spike protein that have been routinely modified in response to selective pressure. An ideal vaccine would avoid targeting these mutation-permissive loci, focusing instead on conserved loci critical for protein function and more likely to resist future variant emergence. Unfortunately, methods to guide antibody responses away from non-critical epitopes are limited. The selection of specific epitope responses for inclusion into the "final" antibody response repertoire is known to be dependent on two critical factors - the precursor frequency of B cells targeting that epitope, and their affinity for the epitope. Unfortunately, these factors cannot be easily leveraged in human vaccination due to the relatively fixed nature of the underlying B cell repertoire. However, limited success has recently resulted from the leveraging of a third factor - rare epitope suppression (RES). RES is a principle of B cell selection whereby B cells that target rare epitopes within a complex antigen pool are selected against, independently of receptor affinity, due to a failure to attract sufficient T cell help. Conversely, B cells that target conserved epitopes within a vaccine pool are more likely to be incorporated in the final humoral response. We hypothesize that RES can be effectively leveraged in the creation of epitope-targeted vaccines. By making an undecided epitope "rare" within a heterologous vaccine pool, it can be de-prioritized in the resulting humoral response. This has been capitalized on by limited studies in influenza using "chimeric" nanoparticles containing multiple strains of hemagglutinin, but the creation of these nanoparticles is technically challenging, and the potency is limited by the number of strains that can be functionally produced and decorated. However, the success of mRNA vaccine technology offers a new way forward. Taking advantage of targeted degenerate nucleotide incorporation into full-length gene synthesis, gene libraries can be generated where specific codons are diversified (or "wobbled") to encode multiple amino acid identities. Cloned into existing mRNA vaccine platforms, WobbleVax libraries will drive the in vivo production of heterogeneous antigen pools with enormous diversity at undesired antigen loci. We expect that selection will favor B cells that avoid these unstable epitopes, focusing instead on conserved regions of the antigen library and driving cross-variant protection. In this study, we propose the generation and testing of a first-generation wobble vaccine using the well-characterized hen egg lysozyme (HEL) system. We will use this simplified model to assess the efficacy of the WobbleVax platform to generate robust humoral immune responses, and importantly, modify epitope-specific response priorities. Specifically, we will make use of a highly characterized mouse model of vaccine immunodominance (the swHEL/HEL vaccine model) to characterize the potential of this new technology in the suppression of robustly immunodominant responses. In parallel, we propose the design and manufacture of a WobbleVax library targeting the SARS-CoV-2 spike protein, wielding the enormous sequence diversity that has been made available through the pandemic into a centralized vaccination library containing more than 109 theoretical members. We will evaluate those libraries against more traditional "fixed" mRNA vaccine approaches that have already been produced, and identify the cross-reactive potential of the WobbleVax platform in driving variant-resistant responses. The design of these vaccine libraries is complex, providing the opportunity to confirm feasibility, optimize design approach, and establish a pipeline for wobble vaccine production and testing in a translatable setting. Altogether, the wobble vaccination platform combines the cutting edge of mRNA vaccination and gene synthesis with new and emerging concepts in viral vaccine targeting. While rooted in sound theoretical footing and well-established methodology, it represents an entirely novel approach to vaccination, and if successful, would have enormous translational potential in the design of target-optimized vaccines capable of driving cross-protective, broadly neutralizing vaccines against many of our most critical human pathogens including SARS-CoV-2, influenza, and HIV. Less