mRNA vaccine-induced IgG mediates nasal SARS-CoV-2 clearance in mice

Abstract

Coronavirus disease 2019 (COVID-19) mRNA vaccines that have contributed to controlling the SARS-CoV-2 pandemic induce specific serum antibodies, which correlate with protection. However, the neutralizing capacity of antibodies for emerging SARS-CoV-2 variants is altered. Suboptimal antibody responses are observed in patients with humoral immunodeficiency diseases, ongoing B cell depletion therapy, and aging. Common experimental mouse models with altered B cell compartments, such as B cell depletion or deficiency, do not fully recapitulate scenarios of declining or suboptimal antibody levels as observed in humans. We report on SARS-CoV-2 immunity in a transgenic mouse model with restricted virus-specific antibodies. Vaccination of C57BL/6-Tg(IghelMD4)4Ccg/J mice with unmodified or N1mΨ-modified mRNA encoding for ancestral spike (S) protein and subsequent challenge with mouse-adapted SARS-CoV-2 provided insights into antibody-independent immunity and the impact of antibody titers on mucosal immunity. Protection against fatal disease was independent of seroconversion following mRNA vaccination, suggesting that virus-specific T cells can compensate for suboptimal antibody levels. In contrast, mRNA-induced IgG in the nasal conchae limited the local viral load and disease progression. Our results indicate that parenteral mRNA immunization can elicit nasal IgG antibodies that effectively suppress local viral replication, highlighting the potential of vaccines in controlling SARS-CoV-2 transmission and epidemiology.

Keywords: IgG; MT: Oligonucleotides: therapies and applications; SARS-CoV-2 MA20; antibodies; antibody-deficient mouse; transmission; upper respiratory tract; viral shedding.

Graphical abstract

Figure 1

WT and IghelMD4 mice exhibit similar SARS-CoV-2-specific T cell responses (A) Study design: female (♀) and male (♂) WT and IghelMD4 mice (n = 162) were primed (day 0) and boosted (day 28) with NaCl (Sham), 8.0 μg N1mΨ-mRNA, or 8.0 μg unmodified mRNA vaccines encoding the S protein of SARS-CoV-2. On day 56, 128 mice (n = 128) were challenged with >10⁴ TCID₅₀ of SARS-CoV-2 MA20 in three independent experiments. Mice were analyzed for anti-SARS-CoV-2 antibodies in nasal tissue and serum and T cell responses in the lungs and spleen before and after the challenge. Survival, weight changes, and viral loads were monitored up to 12 DPI or upon reaching the humane endpoint criteria. Created with BioRender.com. (B–H) Tissue-residing (CD45_IV⁻) T cells from the lungs of mice were analyzed for CD69 (B and E), PD-1 (C and F), CXCR3 (D and G) expression, and frequencies of CD8⁺ effector memory T cells (H). (I and J) Single-cell suspensions obtained from the lungs of mice were restimulated in vitro with S peptides. The percentage of CD8⁺ T cells that expressed granzyme B (I) or IFN-γ (J) in the lungs (day 56) is depicted. The values were obtained by subtracting percentage (unstimulated) from percentage (S peptide stimulated). (B–J) Each data point represents one animal. n = 5 per vaccine and mouse group. n = 6 for Sham WT and N1mΨ-mRNA IghelMD4. The median with the interquartile range is displayed. p values were determined by nonparametric one-way ANOVA and Dunn’s multiple comparison test.

Figure 2

WT and IghelMD4 mice elicit distinct levels of neutralizing antibodies upon mRNA vaccination (A) RBD ELISA of serum from Sham- or mRNA-vaccinated mice pre-challenge. Number of animals per time point: n = 33 for Sham WT, n = 30 for Sham IghelMD4, n = 35 for N1mΨ-mRNA WT, n = 34 for N1mΨ-mRNA IghelMD4, n = 15 for mRNA WT and IghelMD4. The dotted line (…) represents the cutoff for seroconversion (OD = 0.3). The median with the interquartile range is displayed. p values were determined by nonparametric one-way ANOVA and Dunn’s multiple comparison test. (B and C) Statistical analysis of matched RBD ELISA samples. Number of animals per time point: n = 35 for N1mΨ-mRNA WT, n = 34 for N1mΨ-mRNA IghelMD4, n = 15 for mRNA WT and IghelMD4. p values were determined by two-way ANOVA and Šídák’s multiple comparisons test. (D–F) Surrogate virus neutralization test (sVNT) at days 28 and 56 for Sham (D), N1mΨ-mRNA (E), and unmodified mRNA (F). Number of animals per time point: n = 34 for Sham WT, n = 30 for Sham IghelMD4, n = 35 for N1mΨ-mRNA WT, n = 34 for N1mΨ-mRNA IghelMD4, n = 15 for mRNA WT and IghelMD4. Error bars represent the min to max range. p values were determined by two-way ANOVA with Šídák’s multiple comparisons test. The dotted line (…) represents the cutoff for sVNT positivity (30% inhibition) according to the manufacturer’s manual. (A–F) Each data point represents one animal.

Figure 3

mRNA vaccine-induced serum antibody levels determine disease progression without impacting survival (A and B) Survival analysis for Sham, N1mΨ-mRNA, or mRNA vaccinated WT and IghelMD4 mice (A) and IghelMD4 mice vaccinated with N1mΨ-mRNA or mRNA divided by seroconversion status (B) after infection with SARS-CoV-2 MA20 up to 12 DPI. p values were determined by direct curve comparison (Kaplan-Meier) using log rank (Mantel-Cox) test. (A) Number of animals: n = 24 for Sham WT, n = 20 for Sham IghelMD4, n = 23 for N1mΨ-mRNA WT, n = 24 for N1mΨ-mRNA IghelMD4, n = 10 for mRNA WT and IghelMD4. (B) Number of animals: n = 15 for N1mΨ-mRNA no seroconversion, n = 9 N1mΨ-mRNA seroconversion, n = 7 for mRNA no seroconversion, n = 3 for mRNA seroconversion. (C and D) Weight changes of mice infected with SARS-CoV-2 MA20 until 12 DPI for Sham, N1mΨ-mRNA, or mRNA vaccinated WT and IghelMD4 mice (C) and IghelMD4 mice vaccinated with N1mΨ-mRNA or mRNA divided by seroconversion status (D). The dataset passed the D'Agostino-Pearson normality test (α = 0.05). Each data point represents the mean with SEM. p values were determined by two-way ANOVA (mixed effects analysis). (C) Number of animals: n = 28 for Sham WT, n = 24 for Sham IghelMD4, n = 27 for N1mΨ-mRNA WT, n = 28 for N1mΨ-mRNA IghelMD4, n = 10 for mRNA WT and IghelMD4. (D) Number of animals: n = 17 for N1mΨ-mRNA no seroconversion, n = 11 N1mΨ-mRNA seroconversion, n = 7 for mRNA no seroconversion, n = 3 for mRNA seroconversion. (A–D) Exclusion of animals that reached the predetermined study endpoint at 3 DPI (n = 4 per group for Sham and N1mΨ-mRNA) from survival analysis caused differences in the number of animals in survival (A and B) and weight change plots (C and D) for each group.

Figure 4

WT and IghelMD4 mice display distinguishing antibody and T cell responses following SARS-CoV-2 MA20 infection (A–F) Tissue-residing (CD45_IV⁻) T cells from the lungs (A–C) and spleen (D–F) of mice were analyzed at 12 DPI for CD69 (A and D), PD-1 (B and E), and CXCR3 (C and F) expression. Number of animals: n = 10 per vaccine and mouse group, n = 9 for Sham WT (lungs). The median with the interquartile range is displayed. (G) Analysis of RBD ELISA of serum samples from Sham-, N1mΨ-mRNA-, or mRNA-vaccinated mice at 1–6 and 12 DPI. Number of animals: n = 10 (1–6 DPI) and n = 17 (12 DPI) for Sham WT, n = 9 (1–6 DPI) and n = 15 (12 DPI) for Sham IghelMD4, n = 4 (1–6 DPI) and n = 23 (12 DPI) for N1mΨ-mRNA WT, n = 6 (1–6 DPI), and n = 22 (12 DPI) for N1mΨ-mRNA IghelMD4, n = 10 (12 DPI) for mRNA WT and IghelMD4. The dotted line (…) represents the cutoff for seroconversion (OD = 0.3). (A–G) p values were determined by nonparametric one-way ANOVA and Dunn’s multiple comparison test. (H) Surrogate virus-neutralization test (sVNT) at 1–12 DPI for Sham-, N1mΨ-mRNA-, or mRNA-vaccinated WT and IghelMD4 mice. Number of animals: n = 28 for Sham WT, n = 27 for Sham IghhelMD4, n = 27 for N1mΨ-mRNA WT, n = 28 for N1mΨ-mRNA IghelMD4, n = 10 for mRNA WT and IghelMD4. Error bars represent the min to max range. p values were determined by two-way ANOVA with Šídák’s multiple comparisons test. The dotted line (…) represents the cutoff for sVNT positivity (30% inhibition). (A–H) Each data point represents one animal.

Figure 5

Mucosal IgG is required to clear SARS-CoV-2 from the conchae of experimentally infected mice (A) Viral loads in the conchae of SARS-CoV-2 MA20-infected mice on 12 DPI. The detection limit was set at 10³ viral RNA copies per mL, represented by a dotted line (…). Number of animals: n = 17 for Sham WT, n = 15 for Sham IghelMD4, n = 23 for N1mΨ-mRNA WT, n = 22 for N1mΨ-mRNA IghelMD4, n = 10 for mRNA WT and IghelMD4. (B–D) Linear regression and correlation analysis of conchae viral loads with RBD ELISA absorbance (day of death) (B), weight on the day of death for Sham-vaccinated animals (C), and S peptide-specific CD8⁺ IFN-γ⁺ T cells for IghelMD4 mice at 12 DPI (D). (E–H) IgG1 (E and G) and IgG2c (F and H) ELISA of conchae homogenized tissue supernatants on 1–6 (E and F) and 12 DPI (G and H). (E and F) Number of animals: n = 10 for Sham WT, n = 9 for Sham IghelMD4, n = 4 for N1mΨ-mRNA WT, n = 6 for N1mΨ-mRNA IghelMD4. (G and H) Number of animals: n = 17 for Sham WT, n = 11 for Sham IghelMD4, n = 20 for N1mΨ-mRNA WT, n = 17 for N1mΨ-mRNA IghelMD4, n = 5 for mRNA WT, n = 9 for mRNA IghelMD4. (A and E–H) Each data point represents one animal. The median with the interquartile range is displayed. p values were determined by nonparametric one-way ANOVA and Dunn’s multiple comparison test. (B) Number of animals: n = 27 for Sham WT, n = 24 for Sham IghelMD4, n = 27 for N1mΨ-mRNA WT, n = 28 for N1mΨ-mRNA IghelMD4, n = 10 for mRNA WT and IghelMD4. (C) Number of animals: n = 27 for WT, n = 24 for IghelMD4. (D) Number of animals: n = 10 per condition. (B–D) Two-tailed Spearman rank correlation was performed with R (version 4.3.2) in R studio (2023.12.1 build 402) using cor.test(). The regression line was added using geom_smooth() and the linear model (lm) method within the ggplot2 visualization package. The gray area represents the 95% confidence interval.