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Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model
Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model
Major Article
Background. Post-vaccination myopericarditis is reported after immunization with coronavirus disease 2019 (COVID-19)
messenger RNA (mRNA) vaccines. The effect of accidental 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 cy-
tokine/chemokine 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 degener-
ation, apoptosis, and necrosis with adjacent inflammatory cell infiltration and calcific deposits on visceral pericardium, although ev-
idence of coronary artery or other cardiac pathologies was absent. 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. mouse model; SARS-CoV-2; mRNA vaccine; intravenous; COVID-19.
Safe and effective whole-population vaccination against severe (COVID-19) pandemic [1], which has caused about 200 mil-
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the lion cases of COVID-19 globally and over 4 million deaths by
only long-term solution to the ongoing coronavirus disease 2019 17 July 2021[2]. However, the one-dose vaccination rate in the
United States and United Kingdom was only 54.9% and 67.8%,
respectively, as of 10 July 2021[3]. Vaccine hesitancy among the
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.
mice given IV mRNA COVID-19 vaccine can develop acute showed occasional positive cardiomyocytes, infiltrating im-
myopericarditis. Similar histopathological changes and severity mune cells and vascular endothelial cells within the myocar-
were found in male mice (Supplementary Figures 2B, 2C). dium and pericardium in the IV group but not IM vaccine
or NS control groups (Figures 4A, 4B, 4C). Using RT-qPCR
IV mRNA Vaccine Administration Induced Apoptosis of Cardiomyocytes, [12], the amount of COVID-19 mRNA Spike-RBD gene
Tissue mRNA, and Protein Expression of SARS-CoV-2 Spike
copies in heart tissues was significantly higher in IV than IM
To determine if apoptosis was induced in cardiomyocytes,
group at 1 dpi (Supplementary Figure 3). Although no statis-
TUNEL immunofluorescent staining was used for detection of
tically significant differences were found, the mean amount
DNA fragmentation in apoptotic cells (Figure 3A). Apoptotic
of Spike-RBD mRNA was higher in the IV group than the IM
cardiomyocytes were significantly more often found in the IV
group at all other time points.
group than the IM or NS groups. Apoptotic cardiomyocytes dis-
tributed both sporadically or as large foci were found in 75% of IM mRNA Vaccine Administration Only Induced Mild Myocardial
IV group at 1 dpi (6/8) and 38.5% at 2 dpi (5/13) (P < .05; Table Congestion and Edema
1, Figure 3B). No grossly visible change of the heart in the IM group was
To understand whether mRNA vaccine can transfect seen on autopsy or low power scan (Figure 5A). H&E sections
cardiomyocytes to express SARS-CoV-2 spike protein, we showed some myocardial vascular congestion and mild in-
used immunostaining to detect SARS-CoV-2 Spike-RBD and terstitial edema at 1 dpi (Figure 5B). Degenerative changes of
Pathology a mRNA Vaccine Injection (Second Dose Given 14 days After First Dose)
Heart Grossly visible white patches on visceral pericardium 5/13 (38.5%)† 0/6 0/12 3/9 (33.3%) 1/6 (16.7%)
(Fig 1C, 1D)
Pericardial calcific deposit 5/13 (38.5%)† 0/6 0/12 4/9 (44.4%)† 2/6 (33.3%)
(Fig. 2A, 2D)
Pericardial WBC infiltration 9/13 (69.2%)* ,††† 0/6 0/12 5/9 (55.6%)†† 2/6 (33.3%)
(Fig. 2C)
cardiomyocytes were occasionally found at 2 dpi (Figure 5B). higher serum concentrations of cytokines/chemokines at 1
No obvious immune cell infiltration, cardiomyocyte necrosis, dpi, which decreased at 2 dpi (Figure 6C). Increased serum
or TUNEL-positive apoptotic cells were found at 1 or 2 dpi cytokine/chemokine concentrations were found in the IV
(Figure 5C). The changes were not sufficient to satisfy the his- group at 1 dpi, but only CXCL10 and CCL5 were significantly
tological criteria of myocarditis. Notably, SARS-CoV-2 spike higher than the NS control group (Figure 6C). Enzyme im-
protein expression was only found in the infiltrating immune munoassay showed that serum troponin levels of the IV group
cells in the thigh muscle 1 dpi in the IM group. No degenera- (1328.2 ± 325.8 pg/mL) was significantly higher than the IM
tion, necrosis, or SARS-CoV-2 protein expression was evident group (237.5 ± 121.2 pg/mL) and NS group (215 ± 115.9 pg/
in the skeletal myocytes (Figures 5D, 5E). mL) (P < .0001; Table 1).
mRNA Vaccine Administration Induced Inflammatory Cytokine/Chemokine Histopathological Changes of the Heart at 7 dpi and 14 dpi After First Dose,
Response in the Heart, and Increased Serum Troponin and Cytokine/ and at 2 Days After the Second Dose Given at 14 Days After First Dose
Chemokine levels At 7 dpi, the heart of mice in the IV group showed persistent
RT-qPCR assay showed increased mRNA expression of in- changes of myopericarditis (Figure 7A), whereas the IM group
terferon (IFN)-α/β, interleukin (IL)-6, tumor necrosis factor only showed vascular congestion, myocardial edema, and oc-
(TNF)-α, CXCL10, and CCL3 in heart tissue homogenates casional foci of cardiomyocyte degeneration (Figure 7A). At 14
of the IV group, among which IFN-β, IL-6, and TNF-α were dpi, 4/6 (66.7%) of the mice in the IV group showed grossly vis-
significantly higher at 2 dpi than 1 dpi (Figure 6A). As for the ible white patches over the visceral pericardium, and 6/6 (100%)
IM group, all tested inflammatory cytokines/chemokines were showed changes of myopericarditis, compared with only mild
transiently upregulated in heart tissues at 1 dpi and then de- degenerative changes in the IM group (Figure 7B).
creased at 2 dpi (Figure 6B). But IL-1 β expression was signifi- Two days after the second dose of mRNA vaccine, 3/9 (33.3%)
cantly higher in cardiac tissues of male than female mice at 2 dpi and 1/6 (16.7%) of mice in the IM/IV and IV/IM groups devel-
(Supplementary Figure 2A). oped grossly visible white patches over the visceral pericardium,
Beads-based multiplex cytokine/chemokine flow respectively. Both groups showed more diffuse and severe changes
cytometry assay showed that the IM group had significantly of myopericarditis with foci of mild myocardial hemorrhage,
which affected both the right and left heart (Figure 7D; Table 1). and CXCL10 at 2 dpi after IV boosting, which suggested that IV
Moreover, serum cytokine/chemokine levels by beads-based flow vaccine increased inflammatory responses, although CCL2 was in-
cytometry assay showed significantly increased IFN-γ, TNF-α, creased after both IV and IM boosting (Figure 7E).
IV mRNA Vaccine Administration Induced Histopathological Changes subclinical and the changes persisted but did not progress within
in Liver
14 dpi. But these histopathological changes of myopericarditis
H&E-stained sections of the liver tissues of the IV group deteriorated and became rather diffuse after the second dose
showed diffuse ballooning degeneration of hepatocytes and boosting with either IV or IM administration 14 days after the
focal hepatocyte necrosis at 1–2 dpi (Figure 8A), whereas the first dose of priming. Although Balb/c mice have been exten-
liver tissues of the IM group showed much milder changes sively used for modeling myocarditis due to viral, protozoal, and
without hepatocyte necrosis at 1–2 dpi (Supplementary Figure autoimmune insults [15], myocardial mineralization can occur
1B). SARS-CoV-2 spike RBD expression by immunostaining spontaneously with age in inbred laboratory rodents. However,
was occasionally seen in hepatocytes of the IV group but not we have excluded this possibility by the demonstration of highly
the IM group (Figure 8B). Except for some vascular congestion significant differences in the gross pathological and histolog-
in lungs, H&E stained sections of spleen, brain, and kidney at ical changes of myopericarditis between mice challenged by IV
1–2 dpi were unremarkable (Supplementary Figure 1C). versus IM vaccine or NS control (Table 1) [16].
Acute myocardial injury due to hypersensitivity myocarditis
DISCUSSION was reported after smallpox vaccine at a rate of 12.3–463 cases
In a Balb/c mouse model with both male and female mice, IV per 100 000 [17] and very rarely associated with other vaccines
but not IM administration of COVID-19 mRNA vaccine in- for yellow fever and influenza [17–19]. COVID-19 mRNA vac-
duced a rapid onset of multifocal myopericarditis with elevated cines were associated with myopericarditis at a rate of 12.6–24
serum troponin, cardiomyocyte degeneration, and changes of cases per million following a second dose [20]. The clinical
both necrosis and apoptosis, adjacent inflammatory infiltrate of manifestations of acute chest pain, dyspnea, arrhythmia, raised
mononuclear cells, interstitial edema, and visceral pericardial serum troponin, and electrocardiographic and gadolinium en-
calcification within 2 dpi. Moreover, the IL-1β, IFN-β, IL-6 and hanced cardiac magnetic resonance imaging (MRI) changes
TNF-α expression levels generally increased significantly from often started 3–5 days after the second dose of vaccine or oc-
1 dpi to 2 dpi in the IV group but not the IM group. Overall, the casionally after first dose [21, 22]. Two patients without meas-
findings have satisfied the Dallas and immunohistochemical urable SARS-CoV-2 spike immunoglobulin G (IgG) presented
criteria of myocarditis [13]. Similar to findings of cardiac mag- shortly after their first vaccine dose, suggesting that myocardial
netic resonance imaging in human myocarditis, the most prom- damage can happen with just 1 dose of mRNA vaccine as dem-
inent site of focal involvement was the pericardial side of atrial onstrated in our present study [23]. Moreover, the second dose
and ventricular walls [14]. Notably, the myopericarditis was of mRNA vaccine given 14 days after the first dose by either
Figure 3. Representative images of TUNEL (apoptosis biomarker) staining of heart tissues. Groups of mice received COVID-19 mRNA vaccine via intramuscular (IM) or in-
travenous (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× mag-
nification). 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.
other components of mRNA vaccine is unlikely as similar degree and arteries within the reach of a syringe needle in the deltoid
of damage should happen in both IV and IM groups. region are considered too small to allow a rapid IV injection of
Both Pfizer/BioNTech and Moderna have clearly stated vaccine without blowing out the vessel [6]. However, this spec-
that their vaccines should only be given via IM route [30, 31]. ulation also lacks supportive scientific evidence. Another pos-
However, current CDC [6] and WHO guidelines [5] no longer sibility of getting a high blood mRNA vaccine level is the rapid
recommend precautionary measures during IM vaccine admin- movement of the vaccine through the lymphatic system into
istration. Brief aspiration for blood return during intramuscular the venous circulation. Thus changing the vaccine injection site
injection of medication as a preventive measure against acci- from deltoid to the vastus lateralis muscle of lateral mid-thigh
dental IV injection was previously present in most guidelines may reduce the amount of vaccine lipid nanoparticles reaching
[32]. This practice becomes controversial as scientific evidence the venous circulation due to enhanced uptake by the dendritic
of the perceived benefit of this procedure is lacking for IM in- cells and macrophages at the regional inguinal, iliac and para-
jection of vaccine. The CDC Pink Book 2020 [6] and WHO aortic lymph nodes.
2015 position paper [5] have recommended against aspiration We note that Pfizer has conducted in vivo biodistribution
prior to vaccine injection so as to minimize pain [33]. The veins studies of IM-injected [3H]-labeled lipid-nanoparticle (LNP)
mRNA vaccines in rats [34]. There was some accumulation of stem cells to the myocardium by retrograde venous perfusion
the formulation in the heart at 2 dpi, although much lower than was reported to achieve a better myocardial concentration [35].
concentrations in the liver or spleen. No similar tracer study Moreover, smaller mRNA-vaccine lipid-nanoparticles(100nm
was reported when the mRNA vaccine is IV injected. Besides diameter) can be sucked into larger T tubules (diameter
the possibility of delivery to the heart through the systemic ar- >200 nm) of cardiomyocytes during diastole, but not into T tu-
terial system, the IV-injected SARS-CoV-2 mRNA could the- bules of skeletal myocyte (diameter 20–40 nm) [36]. Thus, the
oretically transfect myocardial cells through smaller cardiac T tubule system of cardiomyocytes may concentrate mRNA-
venous system (Thebesian network), which consists of a layer vaccine lipid-nanoparticles like a sponge.
of vascular endothelial cells continuous with endothelium of Interestingly, we also observed ballooning degeneration of
4 cardiac chambers without interference by valves. Delivery hepatocytes especially in our IV group, which is compatible
of pharmacologic therapy, gene therapy, growth factors, and with the heavy distribution of the mRNA vaccine formulation
in the liver of IM injected rats in the bio-distribution study [34] younger age or male gender. Although our male mice had sim-
and microscopic vacuolation of portal hepatocytes in these rats ilar degree of myopericarditis to female mice, a lower vaccine
[37]. There have been anecdotal reports on autoimmune hepa- dose may show up differences in disease susceptibility due to im-
titis following COVID-19 mRNA vaccines in humans although munological differences or ACE2 expression driven by sex hor-
these reports are yet to be confirmed by population-level vac- mone and X chromosome. Although the histological changes
cine adverse effect monitoring systems [38, 39]. Further re- in the heart of IM group did not amount to myopericarditis, we
search into the potential association of COVID-19 mRNA cannot exclude the possibility of frank myopericarditis in indi-
vaccination and autoimmune hepatitis is required. viduals who may be more susceptible to even a slight amount
Limitations of our study included the lack of data in ex- of mRNA vaccine entering the systemic circulation from intra-
plaining the association of post-vaccination myocarditis with muscular injection. COVID-19 mRNA vaccines are safe and
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 con-
gestion (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
effective, whereas post-vaccination myopericarditis is rare and without aspiration, which carries the risk of inadvertent IV in-
self-limiting [4]. Our study indicates that IV injection of vac- jection. Increasing the size of mRNA-vaccine lipid-nanoparticle
cines might partially contribute to this clinical phenotype, thus or decreasing the vaccine dose in normal adolescents to reduce
warranting a reconsideration of the practice of IM injection risks of myopericarditis warrant further investigations. Careful
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 hepato-
cytes (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.