Fluorescence Microscopy Journal Club

Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection

by Pooja Munnilal Tiwari, Daryll Vanover, Kevin E. Lindsay, Swapnil Subhash Bawage, Jonathan L. Kirschman, Sushma Bhosle, Aaron W. Lifland, Chiara Zurla & Philip J. Santangelo

Nature Communications 2018, 9, p. 946

The lung is a critical prophylaxis target for numerous infectious agents, including human respiratory syncytial virus (RSV) and influenza. Targeted delivery of therapeutics to the organ of interest has the potential to minimize systemic toxicity, anti-antibody immune responses, and reduce the amount of drug required to achieve therapeutic levels. Tiwari et al. develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via to prevent RSV infections in vivo. The research presented demonstrates that GPI-anchored mRNA-expressed antibodies are retained on the plasma membrane of transfected cells and that expressed neutralizing antibodies with and without a membrane anchor can prevent infections in vitro and in vivo. The authors developed a modular toolbox to express synthetic, modified mRNA and prevent viral infections in the lung in a two-step process first by expressing whole palivizumab (secreted, termed sPali) in the lung via synthetic mRNA delivery by intratracheal aerosol. Second, the well-characterized glycosylphosphatidylinositol (GPI) membrane anchor sequence was linked from the decay accelerating factor (DAF) to the palivizumab heavy chain mRNA. Anchored palivizumab was termed aPali; this paper’s hypothesis is that cells transfected with aPali would retain the immunoglobulin on the epithelial surface, increasing its concentration in the lung and improving efficacy.

Tiwari et al. sought to delineate the mechanism by which aPali prevented infection of transfected cells. Conventional spinning disk confocal microscopy does not afford the necessary axial resolution to determine if RSV particles are internalized in aPali transfected cells or merely attached to the plasma membrane. To overcome this limitation of conventional optical microscopy, single molecule localization microscopy was utilized via the dSTORM method to image the spatial distribution of aPali. In transfected cells, the researchers observed aPali at the plasma membrane along the contour of the cell, and at various post-infection timepoints, individual RSV virions of ~100-300 nm were observed between 100 and 200 nm above the aPali-labeled membrane. These results revealed that RSV particles were not internalized over time, demonstrating that the mechanism by which aPali inhibits infection is by preventing fusion and cytosolic uptake of RSV. This level of optical verification of the localization of the RSV virions would not be possible with conventional microscopy methods and is only possible through the use of advanced optical techniques such as single molecule localization microscopy.