Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model

Abstract

Background: Post-vaccination myopericarditis is reported after immunization with coronavirus disease 2019 (COVID-19) messenger RNA (mRNA) vaccines. The effect of inadvertent intravenous injection of this vaccine on the heart is unknown.

Methods: We compared the clinical manifestations, histopathological changes, tissue mRNA expression, and serum levels of cytokine/chemokine and troponin in Balb/c mice at different time points after intravenous (IV) or intramuscular (IM) vaccine injection with normal saline (NS) control.

Results: Although significant weight loss and higher serum cytokine/chemokine levels were found in IM group at 1-2 days post-injection (dpi), only IV group developed histopathological changes of myopericarditis as evidenced by cardiomyocyte degeneration, apoptosis, and necrosis with adjacent inflammatory cell infiltration and calcific deposits on visceral pericardium, although evidence of coronary artery or other cardiac pathologies was absent. Serum troponin level was significantly higher in IV group. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen expression by immunostaining was occasionally found in infiltrating immune cells of the heart or injection site, in cardiomyocytes and intracardiac vascular endothelial cells, but not skeletal myocytes. The histological changes of myopericarditis after the first IV-priming dose persisted for 2 weeks and were markedly aggravated by a second IM- or IV-booster dose. Cardiac tissue mRNA expression of interleukin (IL)-1β, interferon (IFN)-β, IL-6, and tumor necrosis factor (TNF)-α increased significantly from 1 dpi to 2 dpi in the IV group but not the IM group, compatible with presence of myopericarditis in the IV group. Ballooning degeneration of hepatocytes was consistently found in the IV group. All other organs appeared normal.

Conclusions: This study provided in vivo evidence that inadvertent intravenous injection of COVID-19 mRNA vaccines may induce myopericarditis. Brief withdrawal of syringe plunger to exclude blood aspiration may be one possible way to reduce such risk.

Keywords: COVID-19; SARS-CoV-2; intravenous; mRNA vaccine; mouse model.

Figure 1.

Schema for vaccine administration and gross pathology of mouse after vaccination. A, Experimental schema. Groups of mice were injected with COVID-19 mRNA vaccine via intramuscular (IM) or intravenous (IV) route. At 1, 2, 7, and 14 dpi, mice were killed for histopathological analysis. Normal saline (NS) was IV or IM injected in parallel as control. B, Body weight changes of mice after injection. C, Representative images of gross pathology of mouse organs and heart at 1 dpi. Hearts of NS control and IM vaccine groups appeared normal, whereas whitish patches (arrows) were seen on the visceral pericardium of hearts after IV vaccine. D, Representative images of gross pathology of mouse organs including heart at 2 dpi. Large whitish patches (arrows) were seen on the visceral pericardium of mice receiving IV vaccine. Abbreviations: COVID-19, coronavirus disease 2019; dpi, days post-injection; mRNA, messenger RNA.

Figure 1.

Schema for vaccine administration and gross pathology of mouse after vaccination. A, Experimental schema. Groups of mice were injected with COVID-19 mRNA vaccine via intramuscular (IM) or intravenous (IV) route. At 1, 2, 7, and 14 dpi, mice were killed for histopathological analysis. Normal saline (NS) was IV or IM injected in parallel as control. B, Body weight changes of mice after injection. C, Representative images of gross pathology of mouse organs and heart at 1 dpi. Hearts of NS control and IM vaccine groups appeared normal, whereas whitish patches (arrows) were seen on the visceral pericardium of hearts after IV vaccine. D, Representative images of gross pathology of mouse organs including heart at 2 dpi. Large whitish patches (arrows) were seen on the visceral pericardium of mice receiving IV vaccine. Abbreviations: COVID-19, coronavirus disease 2019; dpi, days post-injection; mRNA, messenger RNA.

Figure 2.

Representative histopathological images of heart tissues of mice receiving IV vaccine. Groups of mice were given IV injection of COVID-19 mRNA vaccine (IV group) and NS (NS group) as control. At 1 and 2 dpi, mice were killed for histopathology analysis. A, Low magnification microscopic scanning images of heart sections (4× magnification). Mice received IV NS showed no detectable histological changes in the heart. After IV mRNA vaccine, the scan images showed thickened and dark blue stained visceral pericardium on the surface of right atrium and right ventricle at both 1 and 2 dpi (arrows). B, H&E stain of heart tissue of IV NS group showed no histological damage in the myocardium and cardiomyocytes. C, H&E stained heart tissue showed inflammatory infiltrates of the myocardium at 2 dpi. Arrows indicated infiltrates of inflammatory cells at 400× magnification. D, H&E stained heart tissue of IV vaccine group at 2 dpi showing thickened visceral pericardium with clusters of dark blue crystal-like structure which indicated calcific deposits (arrows) with adjacent inflammatory cell infiltrates and cardiomyocytes degeneration at. 400× magnification. E, H&E stained myocardial tissue showing cardiomyocytes degeneration at 2 dpi in IV group as indicated by arrows at 400× magnification. F, H&E image showing cardiomyocytes necrosis (arrows at 400× magnification) with immune cells infiltration in IV group at 2 dpi. G, Images of immunohistochemistry staining of white blood cells marker CD45, CD68, and CD3 in the heart sections, showing myocardial and visceral pericardial infiltration by CD45 positive cells. Immunostaining of macrophage marker CD68 showed many positives in the infiltrating cells, with less frequently positive CD3 biomarker for T lymphocytes. Abbreviations: COVID-19, coronavirus disease 2019; dpi, days post-injection; H&E, hematoxylin and eosin; IV, intravenous; mRNA, messenger RNA; NS, normal saline.

Figure 2.

Representative histopathological images of heart tissues of mice receiving IV vaccine. Groups of mice were given IV injection of COVID-19 mRNA vaccine (IV group) and NS (NS group) as control. At 1 and 2 dpi, mice were killed for histopathology analysis. A, Low magnification microscopic scanning images of heart sections (4× magnification). Mice received IV NS showed no detectable histological changes in the heart. After IV mRNA vaccine, the scan images showed thickened and dark blue stained visceral pericardium on the surface of right atrium and right ventricle at both 1 and 2 dpi (arrows). B, H&E stain of heart tissue of IV NS group showed no histological damage in the myocardium and cardiomyocytes. C, H&E stained heart tissue showed inflammatory infiltrates of the myocardium at 2 dpi. Arrows indicated infiltrates of inflammatory cells at 400× magnification. D, H&E stained heart tissue of IV vaccine group at 2 dpi showing thickened visceral pericardium with clusters of dark blue crystal-like structure which indicated calcific deposits (arrows) with adjacent inflammatory cell infiltrates and cardiomyocytes degeneration at. 400× magnification. E, H&E stained myocardial tissue showing cardiomyocytes degeneration at 2 dpi in IV group as indicated by arrows at 400× magnification. F, H&E image showing cardiomyocytes necrosis (arrows at 400× magnification) with immune cells infiltration in IV group at 2 dpi. G, Images of immunohistochemistry staining of white blood cells marker CD45, CD68, and CD3 in the heart sections, showing myocardial and visceral pericardial infiltration by CD45 positive cells. Immunostaining of macrophage marker CD68 showed many positives in the infiltrating cells, with less frequently positive CD3 biomarker for T lymphocytes. Abbreviations: COVID-19, coronavirus disease 2019; dpi, days post-injection; H&E, hematoxylin and eosin; IV, intravenous; mRNA, messenger RNA; NS, normal saline.

Figure 3.

Representative images of TUNEL (apoptosis biomarker) staining of heart tissues. Groups of mice received COVID-19 mRNA vaccine via intramuscular (IM) or intravenous (IV) route. At 1–2 dpi, the mice were killed for histopathological analysis. Control group of mice received IV NS. A, No TUNEL staining signal in heart tissue section of NS control mice at 2 dpi (left) at 200× magnification. DNase treatment of the same tissue as positive control showed TUNEL staining in the nucleus of cardiomyocytes (green fluorescent signal as indicated by arrows in the insert at 400× magnification). B, At 1–2 dpi of IV mRNA vaccine, TUNEL signals were shown in a large area of the myocardial tissue (circled by dashed lines at 40× magnification, indicated by arrows in the inserts at 400× magnification). H&E staining of the same heart tissue sections are shown on the right. Abbreviations: COVID-19, coronavirus disease 2019; dpi, days post-injection; H&E, hematoxylin and eosin; mRNA, messenger RNA; NS, normal saline; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4.

Immunohistochemical staining for protein expression of spike receptor binding domain (RBD) in heart sections. A, Representative images of immunohistochemical staining of heart section showed occasional spike RBD positive cardiomyocytes (arrows at 400× magnification) at 1 dpi in IV vaccine group. H&E stain of the same area of the section is on the right. B, Representative images showing spike RBD expression in the histiocytes, and vascular endothelial cells in myocardium (arrows in 400× magnification). C, Representative images of positive control for the staining of spike RBD in SARS-CoV-2 infected mouse bronchiolar epithelium (arrows in left panel), negative expression of spike RBD by immunostaining in the myocardium of IV NS control group (middle), and immunostaining with only biotin conjugated secondary antibody in heart sections of IV vaccine group at 1 dpi (right). Abbreviations: dpi, days post-injection; H&E, hematoxylin and eosin; IV, intravenous; mRNA, messenger RNA; NS, normal saline; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 5.

Representative histopathological images of heart tissues after intramuscular administration of mRNA vaccine. Groups of mice were given IM vaccine NS (NS group) as control. At 1–2 dpi, mice were killed for histopathology. A, Low magnification scanning images of heart sections (4× magnification). Both NS group and IM vaccine group showed no histological changes in the heart. B, Representative H&E images of heart tissues. NS groups showed normal myocardium and cardiomyocytes. IM vaccine group showed vascular congestion and mild degree of myocardial edema at 1 dpi. No white blood cell infiltration, cardiomyocyte degeneration, or necrosis was observed. At 2 dpi, vascular congestion was reduced but interstitial edema could still be seen. C, TUNEL staining of heart section showed no positive signal at 2 dpi for IM group. D, H&E images of thigh muscle showed white blood cell infiltration in the connective tissue while the adjacent skeletal muscle cells are unremarkable (magnified image in the right) 1 dpi after IM mRNA vaccine. E, Immunohistochemistry staining of Spike RBD showed only some infiltrating white blood cells expressing spike RBD in thigh muscle of IM group (arrows in magnified images). Abbreviations: dpi, days post-injection; H&E, hematoxylin and eosin; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RBD, receptor binding domain; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 5.

Representative histopathological images of heart tissues after intramuscular administration of mRNA vaccine. Groups of mice were given IM vaccine NS (NS group) as control. At 1–2 dpi, mice were killed for histopathology. A, Low magnification scanning images of heart sections (4× magnification). Both NS group and IM vaccine group showed no histological changes in the heart. B, Representative H&E images of heart tissues. NS groups showed normal myocardium and cardiomyocytes. IM vaccine group showed vascular congestion and mild degree of myocardial edema at 1 dpi. No white blood cell infiltration, cardiomyocyte degeneration, or necrosis was observed. At 2 dpi, vascular congestion was reduced but interstitial edema could still be seen. C, TUNEL staining of heart section showed no positive signal at 2 dpi for IM group. D, H&E images of thigh muscle showed white blood cell infiltration in the connective tissue while the adjacent skeletal muscle cells are unremarkable (magnified image in the right) 1 dpi after IM mRNA vaccine. E, Immunohistochemistry staining of Spike RBD showed only some infiltrating white blood cells expressing spike RBD in thigh muscle of IM group (arrows in magnified images). Abbreviations: dpi, days post-injection; H&E, hematoxylin and eosin; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RBD, receptor binding domain; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 6.

Cytokine/chemokine mRNA expression in heart and their serum levels. At 1–2 dpi of IV or IM mRNA vaccine, organs and serum were collected. A, Cytokines/chemokines mRNA expression in heart homogenates of IV group. B, Cytokines/chemokines mRNA expression in heart homogenates of IM group; mRNA expressions were detected by RT-qPCR with gene specific primers. House-keeping gene β-actin mRNA expression was included to normalize the amount of RNA. Data presented were relative gene expression to NS control mice. Error bars induced mean ± standard deviation. n = 5 in each group. C, Serum cytokine/chemokine concentrations at 1–2 dpi were detected by beads-based multiplex flow cytometer assay. Mice serum from IV and IM injection of 50 µL NS were used as control. Error bars indicated mean ± standard deviation. n = 5 each group. *P < .05, **P < .01, ***P < .001,****P < .0001. Abbreviations: dpi, days post-injection; IFN, interferon; IL, interleukin; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TNF, tumor necrosis factor.

Figure 6.

Cytokine/chemokine mRNA expression in heart and their serum levels. At 1–2 dpi of IV or IM mRNA vaccine, organs and serum were collected. A, Cytokines/chemokines mRNA expression in heart homogenates of IV group. B, Cytokines/chemokines mRNA expression in heart homogenates of IM group; mRNA expressions were detected by RT-qPCR with gene specific primers. House-keeping gene β-actin mRNA expression was included to normalize the amount of RNA. Data presented were relative gene expression to NS control mice. Error bars induced mean ± standard deviation. n = 5 in each group. C, Serum cytokine/chemokine concentrations at 1–2 dpi were detected by beads-based multiplex flow cytometer assay. Mice serum from IV and IM injection of 50 µL NS were used as control. Error bars indicated mean ± standard deviation. n = 5 each group. *P < .05, **P < .01, ***P < .001,****P < .0001. Abbreviations: dpi, days post-injection; IFN, interferon; IL, interleukin; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TNF, tumor necrosis factor.

Figure 7.

Histopathological changes in the heart at 7 and 14 dpi after first dose of IV or IM mRNA vaccine and 2 dpi after second dose of vaccine. Groups of mice were given IV and IM vaccine or NS as control. At 7–14 dpi, mice were killed for histopathology. Another 2 groups of mice were given second dose of IV or IM mRNA vaccine at 14 days after the first priming dose and sacrificed at 2 dpi after the second boosting dose. A, Representative histopathological images of mouse heart at day 7. Top panel consisted of heart sections of IV group, which showed myocardial infiltration by white blood cells (left, arrows), interstitial edema, cardiomyocytes degeneration (middle, arrows) and necrosis (right, arrows). Lower panel consisted of heart sections from IM group, which showed myocardial interstitial edema (left) and myocardial vascular congestion (middle, arrows), with degeneration of a few cardiomyocytes (right, arrows). B, Representative histopathological images of IV and IM group at day 14. Heart in IV group showed persistent changes of cardiomyocyte degeneration, white blood cell infiltration, and foci of necrosis (arrows). C, Heart of mice in IM group showed minimal degeneration and infiltration but no necrosis. D, Representative histopathological images of the heart at 2 dpi after the second boosting dose given on day 14 after the first priming dose. Mouse heart in both IV and IM second dose group showed interstitial edema and diffuse cardiomyocyte degeneration on the left (arrows). Mouse heart in both IM and IV group showed diffuse inflammatory infiltrate, focal hemorrhage and necrosis (arrows, right). E. Serum cytokine/chemokine concentrations at 2 dpi post second dose were detected by beads-based multiplex flow cytometer assay. The NS group was used as control. Error bars indicated mean ± standard deviation. n = 5 each group. n = 9 for IV second dose boost group, n = 6 for IM second dose boost group and NS control group. *P < .05, **P < .01, ***P < .001 by multiple t test. Abbreviations: dpi, days post-injection; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 7.

Histopathological changes in the heart at 7 and 14 dpi after first dose of IV or IM mRNA vaccine and 2 dpi after second dose of vaccine. Groups of mice were given IV and IM vaccine or NS as control. At 7–14 dpi, mice were killed for histopathology. Another 2 groups of mice were given second dose of IV or IM mRNA vaccine at 14 days after the first priming dose and sacrificed at 2 dpi after the second boosting dose. A, Representative histopathological images of mouse heart at day 7. Top panel consisted of heart sections of IV group, which showed myocardial infiltration by white blood cells (left, arrows), interstitial edema, cardiomyocytes degeneration (middle, arrows) and necrosis (right, arrows). Lower panel consisted of heart sections from IM group, which showed myocardial interstitial edema (left) and myocardial vascular congestion (middle, arrows), with degeneration of a few cardiomyocytes (right, arrows). B, Representative histopathological images of IV and IM group at day 14. Heart in IV group showed persistent changes of cardiomyocyte degeneration, white blood cell infiltration, and foci of necrosis (arrows). C, Heart of mice in IM group showed minimal degeneration and infiltration but no necrosis. D, Representative histopathological images of the heart at 2 dpi after the second boosting dose given on day 14 after the first priming dose. Mouse heart in both IV and IM second dose group showed interstitial edema and diffuse cardiomyocyte degeneration on the left (arrows). Mouse heart in both IM and IV group showed diffuse inflammatory infiltrate, focal hemorrhage and necrosis (arrows, right). E. Serum cytokine/chemokine concentrations at 2 dpi post second dose were detected by beads-based multiplex flow cytometer assay. The NS group was used as control. Error bars indicated mean ± standard deviation. n = 5 each group. n = 9 for IV second dose boost group, n = 6 for IM second dose boost group and NS control group. *P < .05, **P < .01, ***P < .001 by multiple t test. Abbreviations: dpi, days post-injection; IM, intramuscular; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 8.

Histopathological changes in the liver at 2 dpi after IV mRNA vaccine injection. A, Representative H&E images of liver sections. Images in the upper panel show the liver section of NS control mice, with normal morphology of evenly distributed cords of hepatocytes. Lower panel consisted of images of liver at 2 dpi in IV group. The liver showed diffuse degeneration of hepatocytes without a clear morphological organization of hepatic cords. A large focus of cell necrosis was circled by the dashed line. Magnified images (400× magnification) of hepatocyte necrosis (middle panel, arrows in magnified box of left bottom panel) and ballooning degenerative changes of hepatocytes (right panel, arrows in magnified box of right bottom panel). B, Images of immunohistochemistry stained spike RBD in liver sections at 2 dpi after IV vaccine. A few RBD positive cells were indicated by arrows in the inserts (400× magnification). Abbreviations: dpi, days post-injection; H&E, hematoxylin and eosin; IV, intravenous; mRNA, messenger RNA; NS, normal saline; RBD, receptor binding domain.