No Evidence for Myocarditis or Other Organ Affection by Induction of an Immune Response against Critical SARS-CoV-2 Protein Epitopes in a Mouse Model Susceptible for Autoimmunity

Figures

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  Figure 1. Evaluation of inflammation after immunization with SARS-CoV-2 SP peptides and peptide pools. A/JOla mice were immunized with 150 µg peptide on days 0, 7, and 14 [SP: SP peptides n = 6–7, TnI n = 10, untreated n = 4; SP pool: SPG peptide n = 6, TnI n = 7, untreated n = 6] (A) Inflammation score of single SP peptide (left) and pooled SP peptide (right) immunized A/JOla mice in percentage based on HE staining scored by two experienced investigators (B) Representative images of paraffin-embedded murine heart tissue sections of 3 µm stained with HE at a 20-fold magnification. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test was performed, and significant values were marked (* p < 0.05 and ** p < 0.01).

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  Figure 2. Evaluation of cardiac damage and dysfunction after immunization with SARS-CoV-2 SP peptide and SP peptide pools. A/JOla mice were immunized with 150 µg peptide on days 0, 7, and 14 [SP: SP peptides n = 6–7, TnI n = 10, untreated n = 4; SP pool: SPG peptide n = 6, TnI n = 7, untreated n = 6]. (A) Determination of hsTnT-levels as a cardiac damage marker in blood serum of SP and SPG peptide immunized mice collected on day 28 [threshold (dot line): 50 pg/mL]. (B) Determination of hsTnT-levels as a cardiac damage marker in blood serum of pooled SP peptide immunized mice collected on day 28. (C) Analysis of cardiac dysfunction via echocardiographic assessment of ejection fraction using M-Mode over a period of 28 days. (D) Antibody production is directed against SPG peptides on day 0 and after a 28-day period. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test (A,B,D) or ordinary two-way ANOVA (C) was performed, and significant values were marked (** p < 0.01, *** p < 0.001).

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  Figure 3. Evaluation of inflammation after immunization with SARS-CoV-2 membrane protein peptides and peptide pools. A/JOla mice were immunized with 150 µg peptide three times [MP34-36 n = 6, TnI n = 10, untreated n = 4; MPG n = 6, TnI n = 7, untreated n = 6]. (A) Inflammation score of single MP peptide (left) and pooled MP peptide (right) immunized A/JOla mice in percentage based on HE staining scored by two experienced investigators (B) Representative images of paraffin-embedded murine heart tissue sections of 3 µm stained with HE at a 20-fold magnification. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test was performed, and significant values were marked (* p < 0.05 and ** p < 0.01).

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  Figure 4. Evaluation of cardiac damage and dysfunction after immunization with SARS-CoV-2 membrane protein peptide and membrane protein peptide pools. A/JOla mice were immunized with 150 µg peptide on days 0, 7, and 14 [MP34-36 n = 6, TnI n = 10, untreated n = 4; MPG n = 6, TnI n = 7, untreated n = 6]. (A) Determination of hsTnT-levels as a cardiac damage marker in blood serum of MP and MPG peptide immunized mice collected on day 28 [threshold (dot line): 50 pg/mL]. (B) Analysis of cardiac dysfunction via echocardiographic assessment of ejection fraction using M-Mode over a period of 28 days. (C) Antibody production directed against MPG peptides on day 0 and after a 28-day period. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test (A,C) or ordinary two-way ANOVA (B) was performed, and significant values were marked (** p < 0.01, *** p < 0.001).

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  Figure 5. Evaluation of inflammation after immunization with SARS-CoV-2 nucleocapsid protein peptides and peptide pools. A/JOla mice were immunized with 150 µg peptide three times [NP30-33 n = 6, TnI n = 10, untreated n = 4; NPG n = 6, TnI n = 7, untreated n = 6]. (A) Inflammation score of single NP peptide (left) and pooled NP peptide (right) immunized A/JOla mice in percentage based on HE staining scored by two experienced investigators (B) Representative images of paraffin-embedded murine heart tissue sections of 3 µm stained with HE at a 20-fold magnification. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test was performed, and significant values were marked (* p < 0.05 and ** p < 0.01).

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  Figure 6. Evaluation of cardiac damage and dysfunction after immunization with SARS-CoV-2 nucleocapsid protein peptide and membrane protein peptide pools. A/JOla mice were immunized with 150 µg peptide on days 0, 7, and 14 [NP30-33 n = 6, TnI n = 10, untreated n = 4; NPG n = 6, TnI n = 7, untreated n = 6]. (A) Determination of hsTnT-levels as a cardiac damage marker in blood serum of NP and NPG peptide immunized mice collected on day 28 [threshold (dot line): 50 pg/mL]. (B) Analysis of cardiac dysfunction via echocardiographic assessment of ejection fraction using M-Mode over a period of 28 days. (C) Antibody production directed against NPG peptides on day 0 and after a 28-day period. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test (A,C) or ordinary two-way ANOVA (B) was performed, and significant values were marked (** p < 0.01, *** p < 0.001).

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  Figure 7. Evaluation of inflammation after immunization with SARS-CoV-2 envelope protein peptides and peptide pools. A/JOla mice were immunized with 150 µg peptide three times [EP37-38n = 6, TnI n = 10, untreated n = 4; EPG n = 6, TnI n = 7, untreated n = 6]. (A) Inflammation score of single EP peptide (left) and pooled EP peptide (right) immunized A/JOla mice in percentage based on HE staining scored by two experienced investigators (B) Representative images of paraffin-embedded murine heart tissue sections of 3 µm stained with HE at a 20-fold magnification. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test was performed, and significant values were marked (* p < 0.05 and ** p < 0.01).

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  Figure 8. Evaluation of cardiac damage and dysfunction after immunization with SARS-CoV-2 envelope protein peptide and membrane protein peptide pools. A/JOla mice were immunized with 150 µg peptide on days 0, 7, and 14 [EP37-38 n = 6, TnI n = 10, untreated n = 4; EPG n = 6, TnI n = 7, untreated n = 6]. (A) Determination of hsTnT-levels as a cardiac damage marker in blood serum of EP and EPG peptide immunized mice collected on day 28 [threshold (dot line): 50 pg/mL]. (B) Analysis of cardiac dysfunction via echocardiographic assessment of ejection fraction using M-Mode over a period of 28 days. (C) Antibody production directed against EPG peptides on day 0 and after a 28-day period. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test (A,C) or ordinary two-way ANOVA (B) was performed, and significant values were marked (** p < 0.01, *** p < 0.001).

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  Figure 9. Evaluation of organ integrity and function after immunization with pooled SARS-CoV-2 peptide pools SPG, NGP, MPG, and EPG. (A) Inflammation score of liver, kidney, lung, intestine, and muscle of immunized A/JOla mice in percentage based on HE staining scored by two experienced investigators. Therefore, the ratio of the inflamed area to the whole section was determined. (B) Investigation of organ function in mice immunized with pooled SARS-CoV-2 peptide sequences. Detection of urea, albumin, creatinine, and AST in blood serum on day 28. (C) Histopathological analysis of the liver and kidney in 20× magnification. 3 µm tissue sections were paraffin-embedded and stained with HE. All values are indicated as mean ± SEM. For statistics, a Mann–Whitney test (A,B) was performed, and significant values were marked (* p < 0.05).

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  Figure 10. Representative images of lung, intestine, and muscle after immunization with SARS-CoV-2 peptide pools SPG, NPG, MPG, and EPG at 20× magnification. 3 µm tissue sections were paraffin-embedded and stained with HE.