The investigation of mRNA vaccines formulated in liposomes administrated in multiple routes against SARS-CoV-2

Abstract

COVID-19 pandemic has resulted in an unprecedented global public health crisis. It is obvious that SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Since obvious advantages including fast manufacturing speed, potent immunogenicity and good safety profile, six mRNA vaccines have been used to prevent SARS-CoV-2 infections in clinic with lipid nanoparticles (LNP) formulation via intramuscular injection. In this work, we first constructed RBD-encoding mRNA (RBD-mRNA) formulated in liposomes (LPX/RBD-mRNA) and investigated the influence of administration routes on the immunogenicity. LPX/RBD-mRNA can express RBD in vivo and successfully induced SARS-CoV-2 RBD specific antibodies in the vaccinated mice, which efficiently neutralized SARS-CoV-2 pseudotyped virus. Moreover, the administration routes were found to affect the virus neutralizing capacity of sera derived from the immunized mice and the types (Th1-type and Th2-type) of cellular immune responses. This study indicated that liposome-based RBD-mRNA vaccine with optimal administration route might be a potential candidate against SARS-CoV-2 infection with good efficacy and safety.

Keywords: Administration route; Liposomes; Receptor-binding domain; SARS-CoV-2; mRNA vaccines.

Graphical abstract

Fig. 1

Preparation and characterization of RBD-mRNA loaded liposome (LPX/RBD-mRNA). (A) Schematic of the RBD-mRNA construct. (B) Sketch map of the preparation of LPX/RBD-mRNA by a thin-film dispersion method. (C) The size distribution of LPX/RBD-mRNA measured by DLS. (D) Typical TEM image of LPX/RBD-mRNA (scale bar 100 nm).

Fig. 2

The time dependent size variation of LPX/RBD-mRNA stored under different conditions.

Fig. 3

The efficient expression of mRNA loaded into LP in vitro. (A) The RBD-mRNA can be successfully translated into RBD protein in HEK293T cells at 24 h post-transfection. (B) Flow cytometry analysis of GFP positive rate of HEK293T and DC2.4 cells at 24 h after transfection with LPX/RBD-EGFP-mRNA. (C) The ELISA detection of RBD expressed in HEK293T cell lysate and secreted into the culture supernatant at 12 h and 24 h after LPX/RBD-mRNA transfection.

Fig. 4

The effective expression of mRNA encapsulated into LPX in vivo (n = 3). (A) The representative image of the expression and biodistribution of FLuc-mRNA at 6 h in C57BL/6 mice treated by LPX/ FLuc-mRNA via intravenous injection. (B) The RBD levels in sera in C57BL/6 mice treated by LPX/RBD-mRNA via different injection routes. * P < 0.05.

Fig. 5

Immune responses induced by LPX/RBD-mRNA in vaccinated C57BL/6 mice (n = 6). (A) Schematic diagram of immunization and sample collection. (B-E) The SARS-CoV-2 RBD specific IgG and IgM antibody titers on day 15 and day 30 determined by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

IgG1 and IgG2a levels in C57BL/6 mice vaccinated with LPX/RBD-mRNA via different injection routes (n = 6). The IgG subtypes in serum on day 30 were determined by ELISA. Statistical analyses were performed using GraphPad Prism 8.01 software. One-way analysis of variance (ANOVA) was used to assess the significance of differences among groups.

Fig. 7

SARS-CoV-2 pseudovirus neutralization. The NT₅₀ titers of the sera on day 30 were determined using SARS-CoV-2 pseudovirus infection. Statistical analyses were performed using GraphPad Prism 8.01 software. One-way analysis of variance (ANOVA) was used to assess the significance of differences among groups.

Fig. 8

Immune tolerability of LPX/RBD-mRNA in the treated mice. The serum concentrations of cytokines including eotaxin, GRO-α, IP-10, RANTES, and MCP-1 were measured by ELISA 12 h after a single administration of 30 μg LPX/RBD-mRNA (n = 3 per group).

Fig. 9

H&E analyses of major organs after boost vaccination with 30 μg LPX/RBD-mRNA (scale bar 200 μm).