Poster CC8 - Hitchhiking across the mucosa: Investigating FcRn-mediated transmucosal uptake of diverse antigens with amphiphile-conjugate subunit vaccines

Introduction: The current clinical standard for vaccine delivery through parenteral administration is not sufficient to elicit immune responses at mucosal portals of entry for many infectious pathogens, such as HIV, SARS-CoV-2, and influenza. These are among the many pathogens that are transmitted via mucosal routes and are thought to require the engagement of both systemic and mucosal immunity for effective management and protection. However, subunit vaccines have historically faced challenges of poor mucosal uptake leading to weak vaccine-induced immune responses. To address this challenge, our lab has engineered a subunit vaccine platform consisting of protein antigen conjugated via polyethylene glycol linker to an albumin-binding lipid tail. ‘Amphiphile vaccines’ overcome conventional challenges with transmucosal uptake by binding to endogenous albumin in the mucus layer and, much like hitchhiking, traverse the mucous membrane via the neonatal 'Fc' receptor (FcRn) that is responsible for transcytosis and recycling of albumin.
The ultimate goal of this work is to explore the molecular design parameters of the amphiphile vaccine platform (such as antigen molecular weight (MW) and linker length) and the role these properties play on the cellular mechanism, trafficking behavior, and activation of systemic and mucosal immune responses. Amphiphile vaccines can adopt three molecular conformations that we hypothesize are dependent on their molecular properties: 1) albumin-bound and monovalent in the presence of albumin; 2) multivalent micelle formation in the absence of albumin; and 3) multivalent ‘painting’ of cells with antigen via membrane insertion of the lipid tail.

Materials and

Methods: The amphiphile vaccine platform was synthesized by covalently conjugating DSPE-PEG-maleimide with a cysteine-terminal protein. For this, HIV peptide/protein antigens of different MW cutoffs were used because they help study the B cell activation in a glVRC01 antigen-specific B cell line. The maleimide group is exchanged with the protein yielding the amph-PEG-protein conjugate. Synthesized amphiphile conjugates were purified through a size-exclusion chromatography (SEC) column. Tryptophan fluorescence of the collected fractions was then measured at (ex 280, em 340) on a plate reader to determine micelle forming conjugate and free protein peak. UV-Vis spectral analysis and dynamic light scattering (DLS) were used to characterize the conjugation, concentration, and particle size of these amphiphile conjugates.
To evaluate membrane insertion, amph-conjugates were labeled with AF647 fluorophore while HIV env antigen-specific VRC01 antibody was labeled with AF555 fluorophore. Amph-conjugates ranging from 25-300 nM concentration were incubated with Ramos B cells for 1 hour at 37C, washed, then ‘stained’ with AF555-VRC01, which would bind to eOD antigen ‘painting’ the cell surface through membrane-insertion of its amphiphile tail. Flow cytometry was used to measure AF647+AF555+ double positive cells to quantify membrane insertion
Next, a calcium flux assay was performed in an engineered glVRC01 B cell line that expresses BCRs specific for HIV env antigens (such as eOD) in order to quantify the effect of vaccine molecular properties and multivalency on B cell activation. In this experiment, Amphiphile conjugates ranging from 25-300 nM concentration were added to glVRC01 cells pre-stained with Fluo-8 Ca2+-binding fluorophore.

Results, Conclusions, and Discussions: Fluorescence assay and DLS unveiled the amphiphilic nature of the conjugate platform. DLS data further shows that amph-eOD can conveniently form stable micelles in PBS anywhere above the critical micellar concentration of ~11.5nM, whereas heavier proteins like amph-MD39 cannot form micelles because of strong steric hindrance by the protein structure. Next, immunofluorescence staining with incubated conjugates revealed that, unlike soluble eOD, amph-eOD was able to undergo successful micelle dissociation in the presence of albumin and exhibited lipid tail insertion into the cell membrane. Furthermore, flow cytometry confirmed that percentage insertion increased with higher antigen concentration conjugated on the platform. This percentage was significantly reduced when heavier molecular weight antigen was introduced which could be possibly because of strong steric hindrance by the protein structure or structural instability caused by increased chain length. Lastly, calcium flux data revealed that both micellar amph-conjugates and membrane-painted amph-conjugates are capable of antigen-specific BCR-mediated activation of gLVRC01 B cells due to their multivalent antigen presentation in contrast to soluble antigens.
From the results observed in this study, we conclude that the conjugation of proteins with optimal molecular weight onto this amphiphile platform can form stable micelles. This is an important property of these conjugates as they can multivalently activate B cells, which we hypothesize plays a role in activating germinal center B cell responses in lymphoid organs and enhancing humoral immune responses. In addition to forming micelles, this structure undergoes a critical transformation upon binding albumin, reverting to its original monomeric chain-like configuration that leads to efficient transmucosal uptake into MALT. This reverted structure is capable of inserting its lipophilic tail into cells for multivalent antigen presentation. Furthermore, this trend was not observed with heavier MW proteins like HIV MD39 trimer conjugates. Lastly, calcium flux results highlighted the importance of multivalency in enhancing B cell activation, showcased by both micelle forming and membrane-inserted amph-conjugates. Upon activation, B cell activation is crucial for immune system activation. Future work will evaluate transmucosal uptake and in vivo immune response.

Acknowledgements (Optional): This work was supported by the resources and staff at the University of Minnesota University Imaging Centers (UIC). SCR_020997. We would also want to acknowledge the support of Kathryn Jans, an undergraduate student in the lab.