Intranasal Immunization with Liposome-Displayed Receptor-Binding Domain Induces Mucosal Immunity and Protection against SARS-CoV-2

Abstract

The global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to efforts in developing effective vaccine approaches. Currently, approved coronavirus disease 2019 (COVID-19) vaccines are administered through an intramuscular (I.M.) route. Here, we show that the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD), when displayed on immunogenic liposomes, can be intranasally (I.N.) administered, resulting in the production of antigen-specific IgA and antigen-specific cellular responses in the lungs. Following I.N. immunization, antigen-presenting cells of the lungs took up liposomes displaying the RBD. K18 human ACE2-transgenic mice that were immunized I.M or I.N with sub-microgram doses of RBD liposomes and that were then challenged with SARS-CoV-2 had a reduced viral load in the early course of infection, with I.M. achieving complete viral clearance. Nevertheless, both vaccine administration routes led to full protection against lethal viral infection, demonstrating the potential for the further exploration and optimization of I.N immunization with liposome-displayed antigen vaccines.

Keywords: RBD; SARS-CoV-2; intranasal; liposomes; vaccines.

Figure 1

Immunogenic liposome-displayed RBD for I.M. and I.N. immunization. K18 hACE2 transgenic mice were immunized with CP/RBD on day 0 and 14 intranasally (I.N.; blue) or intramuscularly (I.M.; red). Lung homogenates and serum were collected on day 28. (A) Schematic representation of the immunization schedule. Anti-RBD lung IgA (B), lung IgG (C) and sera IgG (D) ELISA titers in K18 hACE2 transgenic mice immunized I.N. or I.M. with the indicated RBD dose. Antibody function was assessed with a sVNT assay with post-immune lung homogenates (E) and sera (F). n = 4 mice per group. Lines represent geometric (titer) and arithmetic (sVNT) mean. A two-sided Student T-test using log-transformed titer or sVNT data was used to analyze differences, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Cellular responses induced by CP/RBD immunization. K18 hACE2 transgenic mice were immunized with CP/RBD on day 0 and 14. Lung and splenocytes were collected and restimulated with the RBD. (A) Images of ELISpot results from lungs (top) and spleen (bottom). (B) Quantification of results shown in (A). The lines in (B) represent mean, and two-sided Student T-test using log-transformed data was used to analyze differences, * p < 0.05, ** p < 0.01. Lung and spleen cells from n = 5 mice per group were pooled, and the samples were performed in triplicate.

Figure 3

Uptake of antigen particles into immune cells of the lungs. K18 hACE2 transgenic mice were I.N. administered liposomes also containing CoPoP, PoP (for fluorescence detection) and 3D6A-PHAD with or without RBD display. Two days later, lungs were collected, and liposomal uptake was assessed with flow cytometry. (A) Dendritic cells were gated with CD11c-positive cells and macrophages were gated with F4/80-positive cells. (B) The number of cells with PoP fluorescence in indicated cell type. n = 4 mice per group. The lines in C represent mean, and two-sided Student T-test using log-transformed data was used to analyze differences, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Protection of K18-hACE2 transgenic mice against lethal SARS-CoV-2 challenge following I.N. or I.M. vaccination with CP/RBD. K18-hACE2 transgenic mice (n = 12 per group) were immunized I.M. or I.N. with CP/RBD (0.5 µg RBD) on days 0 and 14 prior to challenge on day 28 with 10⁵ PFU of SARS-CoV-2 WA-1. Mock-vaccinated and mock-infected K18-hACE2 transgenic mice were included as controls. (A) Schematic representation of the immunization schedule and challenge. On day 2 and day 4 post-infection, a cohort of 4 mice was euthanized, lungs (B) and nasal turbinates (C) were collected and homogenized, and viral titers were determined by standard plaque assay. The dotted line shows the limit of detection of the assay. “ND” is not detected. Mice were monitored post-challenge for 14 days for body weight loss (D) and survival (E). Student t-test was used to determine significance between viral titers, *** p < 0.005.