Shallow, L. | bioRxiv Science

bioRxiv2026

Enhancement of a STING Agonist Vaccine for Tuberculosis Using Locally Supercharged MS2 Viral Capsids

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

immunology↗

bioRxiv2022

Cas9+ conditionally immortalized neutrophil progenitors as a tool for genome wide CRISPR screening for neutrophil differentiation and function

Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Murine neutrophil progenitors that are immortalized with estrogen-regulated expression of Hoxb8 differentiate into neutrophils upon withdrawal of estrogen and facilitate the quantitative production of neutrophils in vitro. Here we report the creation of a Cas9+ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils for the first time. By editing progenitors via transduction with sgRNAs, and then withdrawing estrogen, Cas9 edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both in vitro in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9+ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes both negatively and positively selected under differentiation conditions. One of the top hits from this screen was Cebpe, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using the progenitor cell line, we also confirmed that Cepbe is required for neutrophil differentiation in vivo, validating the utility of this cell line both for screening and for studying in vivo phenotypes. The genome-wide screen also identified all components of the WASH complex as being required for neutrophil differentiation, a finding that extends the known role of WASH in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, we demonstrate that Cas9+ER-Hoxb8 immortalized neutrophils can be used to study neutrophil function both in vitro and in vivo. This new resource will enable the analysis of the role of neutrophils in numerous disease states using genetics for the first time.

immunology↗