Effect of Anti-PEG Antibody on Immune Response of mRNA-Loaded Lipid Nanoparticles

Abstract

Lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccines have been approved for use to combat coronavirus disease 2019 (COVID-19). The mRNA-LNPs contain PEG-conjugated lipids. Clinical studies have reported that mRNA-LNPs induce the production of anti-PEG antibodies, but the anti-PEG antibodies do not affect the production of neutralizing antibodies. However, the detailed influence of anti-PEG antibodies on mRNA-LNP vaccines remains unclear. Therefore, in this study, we prepared ovalbumin (OVA) as a model antigen-encoding mRNA-loaded LNP (mRNA-OVA-LNP), and we determined whether anti-PEG antibodies could affect the antigen-specific immune response of mRNA-OVA-LNP vaccination in mice pretreated with PEG-modified liposomes to induce the production of anti-PEG antibodies. After intramuscular (i.m.) injection of the mRNA-LNP, the anti-PEG antibodies did not change the expression of protein or induction of cytokine and cellular immune response but did slightly increase the induction of antigen-specific antibodies. Furthermore, repeated mRNA-LNP i.m. injection induced the production of anti-PEG IgM and anti-PEG IgG. Our results suggest that mRNA-LNP induces the production of anti-PEG antibodies, but the priming of the antigen-specific immune response of mRNA-LNP vaccination is not notably affected by anti-PEG antibodies.

Keywords: anti-PEG antibody; lipid nanoparticle; mRNA; polyethylene glycol; vaccine.

Figure 1

Effects of pre-existing anti-PEG antibody on luciferase expression after i.m. injection of mRNA-Luc-LNP. The PEG-LP was i.v. injected into the mouse. After 7 days, mRNA-Luc-LNP (0.5 μg of mRNA) was i.m. injected into the right leg. After 1 day, the muscles in left and right legs were collected and luciferase expression was evaluated (A). The liver and spleen were also collected and luciferase expression was determined (B). Data are expressed as mean ± SD (n = 6). *P < 0.05.

Figure 2

Effect of pre-existing anti-PEG antibody on cytokine production after mRNA-OVA-LNP administration. The PEG-LP was i.v. injected into the mouse on day −7. The mRNA-OVA-LNP (0.5 μg mRNA) was i.m. injected into the right leg on days 0 and 21. At 6 h after the second administration, plasma was collected, and the cytokine levels were measured. Data are expressed as the mean ± SD (n = 5–6). *P < 0.05.

Figure 3

Effect of pre-existing anti-PEG antibody on the production of OVA-specific antibodies after mRNA-OVA-LNP administration The PEG-LP was i.v. injected into the mouse on day −7. The mRNA-OVA-LNP (0.5 μg mRNA) was i.m. injected into the right leg on days 0 and 21. On day 28, plasma was collected, and OVA-specific IgG, IgG1 and IgG2c were measured through ELISA. Plasma samples with 4,000- (●), 20,000- (■), and 100,000-fold (▲) dilution were used. Data are expressed as mean ± SD (n = 5–6). *P < 0.05.

Figure 4

Effect of pre-existing anti-PEG antibody on the IFN-γ production of splenocyte after mRNA-OVA-LNP administration. The PEG-LP was i.v. injected into the mouse on day −7. The mRNA-OVA-LNP (0.5 μg mRNA) was i.m. injected into the right leg on days 0 and 21. On day 28, splenocytes were collected and stimulated with an OVA or SL8 for 3 days. The production of IFN-γ was determined through ELISA. Data are expressed as the mean ± SD (n = 5–6).

Figure 5

Effect of PEG-LP and mRNA-OVA-LNP on the production of anti-PEG IgM and anti-PEG IgG. The PEG-LP was i.v. injected into the mouse on day −7. The mRNA-OVA-LNP (0.5 μg mRNA) was i.m. injected into the right leg on days 0 and 21. On day 28, plasma was collected, and anti-PEG IgM and anti-PEG IgG were determined via ELISA. Plasma samples with 100- (●), 500- (■), and 2,500-fold (▲) dilutions were used. Data are expressed as mean ± SD (n = 5–6).

Figure 6

Production of anti-PEG IgM and anti-PEG IgG after mRNA-OVA-LNP administration. mRNA-OVA-LNP (0.5 μg mRNA) was i.m. injected into the right leg once on day 0 (A, B) or two times on days 0 and 21 (C, D). On days 0, 7, 14, 21, 28, 35, and 42, plasma was collected, and anti-PEG IgM and anti-PEG IgG were determined via ELISA. Plasma samples with 100- (●), 500- (■), and 2,500-fold (▲) dilutions were used. Data are expressed as mean ± SD (n = 5–6). *P < 0.05 vs other days.