Intraduodenal Delivery of Exosome-Loaded SARS-CoV-2 RBD mRNA Induces a Neutralizing Antibody Response in Mice

Abstract

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has presented numerous challenges to global health. Vaccines, including lipid-based nanoparticle mRNA, inactivated virus, and recombined protein, have been used to prevent SARS-CoV-2 infections in clinics and have been immensely helpful in controlling the pandemic. Here, we present and assess an oral mRNA vaccine based on bovine-milk-derived exosomes (milk-exos), which encodes the SARS-CoV-2 receptor-binding domain (RBD) as an immunogen. The results indicate that RBD mRNA delivered by milk-derived exosomes can produce secreted RBD peptides in 293 cells in vitro and stimulates neutralizing antibodies against RBD in mice. These results indicate that SARS-CoV-2 RBD mRNA vaccine loading with bovine-milk-derived exosomes is an easy, cheap, and novel way to introduce immunity against SARS-CoV-2 in vivo. Additionally, it also can work as a new oral delivery system for mRNA.

Keywords: SARS-CoV-2; bovine-milk-derived exosomes; mRNA; neutralizing antibodies; oral vaccines; receptor-binding domain.

Figure 1

Identification of bovine-milk-derived exosomes in fractions of density gradient ultracentrifugation. (A) Schematic representation of the major steps involved in isolating exosomes from bovine raw milk. (B) F1–F6 represents the corresponding concentrations of sucrose. (C) The exosome suspension was analyzed via Western blot. Immunoblots showed exosome markers in different milk exosome fractions. +, positive control (the protein of HaCat cells); −, negative control (the protein of Hela cells). (D) The morphologies of different fractions obtained via TEM. Scale bars = 500 nm.

Figure 2

Characterization of DC-milk-exos. (A) The morphology and (B) the particle size analysis were detected via TEM and nanoFCM, respectively. (C) A three-way Venn diagram of proteins from three batches of DC-milk-exos revealed 1022 proteins common to all datasets. Cluster analyses for vital exosomal membrane markers CD9, CD63, CD81, and TSG101, microvesicle surface markers GM130, and endoplasmic reticulum (ER) marker calnexin are indicated in the table. Abbreviations: TEM, transmission electron microscope; DC-milk-exos, bovine-milk-derived exosomes via density gradient ultracentrifugation. Scale bars = 500 nm.

Figure 3

Characterization of SARS-CoV-2 receptor-binding domain (RBD) mRNA. (A) Gel-like image of in vitro synthesized RBD mRNA interrogated using an RNA chip on an Agilent Bioanalyzer. Data for RNA markers, RBD mRNA, are presented from left to right. (B) Western blot for SARS-CoV-2 RBD protein expression in 293T cells when the RBD mRNA was transfected into 293T cells with an additional 24 h of treatment along with 1 μg and 3 μg of RBD mRNA. Lanes 1–3: control; Lanes 4–6: 1 μg of RBD-293T; Lanes 7–9: 3 μg of RBD-293T.

Figure 4

Characterization of milk-derived exosome—based vaccine for SARS-CoV-2. (A) Flowchart of vaccine preparation for SARS-CoV-2. The morphology (B), particle size distribution (C), zeta potential (D), and RBD mRNA loading efficiency (E) were determined. Scale bars = 500 nm.

Figure 5

mRNA-loaded exosomes deliver functional SARS-CoV-2 RBD mRNA to human cells in vitro. (A) Western blot analysis of the expression of RBD mRNA delivered by oral vaccine in 293T cells. (B) The ELISA detection of RBD expressed in 293T cell lysate and secreted into the culture supernatant 24 h after the milk-derived exosome-based vaccine transfection. The data are presented as mean standard deviation with a group size of three. ** p < 0.01 vs. PBS.

Figure 6

Validation of SARS-CoV-2 RBD mRNA-loaded milk exosomes delivering functional SARS-CoV-2 RBD mRNA in vivo. (A) Mouse immunization and sera sampling schedule. BALB/c mice received the same doses of an oral vaccine for the SARS-CoV-2—based milk-derived exosomes (N = 5) or the control saline (N = 3) on day 0 and were boosted again on days 14 and 35. Sera were collected on days 0 (pre-vaccination), 7, 14, 21, 28, 35, 42, and 49 (post-vaccination). The blue and red arrows represent the time points of immunization and blood collection, respectively. (B) Preliminary assessments for neutralizing antibodies of the oral vaccine for SARS-CoV-2—based milk-derived exosomes in serum were determined via ELISA. The red dotted line represents the cutoff value of neutralizing antibodies against the RBD peptide. N = 3 (control), and N = 5 (RBD-DC-milk-exos), and the data are presented as mean ± STD. ** p < 0.01, *** p < 0.001 vs. control group.