University of Minnesota - Twin Cities, United States
Introduction: There have been nearly 800 million cases and 7 million deaths worldwide due to COVID-19. Furthermore, lower respiratory infections were the fourth leading cause of death in 2020. To combat persisting and emerging threats from SARS-CoV-2 and other mucosally transmitted pathogens, immunization strategies are needed that elicit immune protection at mucosal portals of entry to better block infection and transmission. While traditional parenteral vaccines typically elicit poor mucosal immunity, mucosal vaccines are known to promote immunity at barrier tissues through initiation of immune responses in underlying MALT. Antigen delivery to MALT can drive programming of mucosa-specific lymphocyte function and mucosal tissue homing. Although well-motivated by the biology of mucosal immunity, the delivery of vaccine components across mucosal barriers is a major challenge for mucosal vaccine development. To overcome this obstacle, we recently developed a strategy of ‘albumin hitchhiking’ across mucosal barriers using the neonatal Fc receptor (FcRn), broadly expressed on mucosal epithelial cells, as a ‘shuttle’. Modifying protein antigens with an amphiphile tail (‘amph-vaccines’) significantly increases uptake across the nasal mucosa through lipophilic binding to endogenous albumin, which is then transcytosed across mucosal epithelium via FcRn.
Materials and
Methods: BALB/C mice were primed intranasally on day 0 and boosted on day 28 with a 5 ug dose of RBD (amph-RBD or soluble RBD) combined with 5 ug of saponin monophosphoryl lipid A nanoparticles (SMNP). Serum and fecal samples were collected bi-weekly and ELISAs were performed to quantify IgG and IgA titers. High binding plates were coated directly with 2ug/ml of RBD antigen in PBS overnight at 4°C to capture antigen specific IgG and IgA antibodies. Serum and fecal samples were diluted 100x and 10x in block buffer (PBS + 2% BSA) respectively, followed by a 4x serial dilution and incubated for 2 hours at room temperature. Goat anti-mouse IgG horseradish peroxidase (HRP) and goat anti-mouse IgA were diluted 5000x and 2000x respectively in block buffer and incubated for 1 hour at room temperature. Plates were developed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate for 5-20 minutes followed by 2N sulfuric acid to stop. The absorbance was measured on a plate reader (A450/A340). Antibody titers were found by calculating the dilution factor at which the log10 of absorbance drops below a cutoff of 0.2 for IgG and 0.1 for IgA.
Results, Conclusions, and Discussions: Mice immunized with amph-RBD showed up to 1,000,000- and 1,000-fold higher serum and fecal IgG titers, respectively, compared to soluble protein over 17 weeks (Figure 1A and B). In addition, mice immunized with amph-RBD showed up to 10,000- and 1,000-fold higher serum and fecal IgA titers, respectively, when compared to soluble protein (Figure 1C and D). We hypothesize that the dip in titers at week 8 is likely due to degradation because of prolonged storage prior to ELISA analysis. Fecal samples are used as a surrogate marker for mucosal immunity at disease relevant sites because many analyses of respiratory mucosal protection require endpoint analysis while fecal sampling is longitudinal. Thus, significant IgA titers in these mucosal samples show evidence of robust mucosal humoral immunity. Continuing studies include investigation of humoral IgG and IgA responses in the upper and lower respiratory mucosa, clinically relevant sites as the portals of entry for SARS-CoV-2. We are also investigating the role of antigen delivery to the NALT and NALT kinetics for establishment of mucosal humoral immunity in the respiratory mucosa. These results suggest that employing amph vaccines to deliver antigen across nasal epithelium is a promising strategy to promote mucosal immunity.
