Longitudinal dynamics of SARS-CoV-2-specific cellular and humoral immunity after natural infection or BNT162b2 vaccination

Abstract

The timing of the development of specific adaptive immunity after natural SARS-CoV-2 infection, and its relevance in clinical outcome, has not been characterized in depth. Description of the long-term maintenance of both cellular and humoral responses elicited by real-world anti-SARS-CoV-2 vaccination is still scarce. Here we aimed to understand the development of optimal protective responses after SARS-CoV-2 infection and vaccination. We performed an early, longitudinal study of S1-, M- and N-specific IFN-γ and IL-2 T cell immunity and anti-S total and neutralizing antibodies in 88 mild, moderate or severe acute COVID-19 patients. Moreover, SARS-CoV-2-specific adaptive immunity was also analysed in 234 COVID-19 recovered subjects, 28 uninfected BNT162b2-vaccinees and 30 uninfected healthy controls. Upon natural infection, cellular and humoral responses were early and coordinated in mild patients, while weak and inconsistent in severe patients. The S1-specific cellular response measured at hospital arrival was an independent predictive factor against severity. In COVID-19 recovered patients, four to seven months post-infection, cellular immunity was maintained but antibodies and neutralization capacity declined. Finally, a robust Th1-driven immune response was developed in uninfected BNT162b2-vaccinees. Three months post-vaccination, the cellular response was comparable, while the humoral response was consistently stronger, to that measured in COVID-19 recovered patients. Thus, measurement of both humoral and cellular responses provides information on prognosis and protection from infection, which may add value for individual and public health recommendations.

Fig 1. Overview of patient cohorts including the time of sample collection.

(A) Patients with natural infection classified according to their maximum disease severity. As depicted, samples were obtained upon arrival to the emergency room (ER), a week after hospitalization (Hosp.), and during the convalescent and recovered phases depending on each cohort. (B) Healthy controls SARS-CoV-2 uninfected with negative PCR and/or serology. (C) Vaccinated individuals who received two doses of BNT162b2 21 days apart. Samples were collected pre-vaccine, pre-boost and 15, 30 and 90 days after receiving the second vaccine dose. Out.: out-patients; In.: in-patients; ICU: Intensive care unit; PaO₂/FiO₂: Partial pressure of arterial oxygen/Fraction of inspired oxygen ratio; PSO: Post-symptom onset. Fig 1 created with BioRender.com.

Fig 2. Cellular and humoral immune responses in mild natural infection, during acute and convalescent phases.

(A) FluoroSpot IFN-γ responses against SARS-CoV-2 S1, M and N proteins according to the days post-symptom onset (DPSO). Data is represented as spot forming unit (sfu) per million PBMC. Dashed lines represent the positivity cut-off established by using a non-infected control group: >25 sfu/10⁶ PBMC for S1, >21 sfu/10⁶ PBMC for M and >14 sfu/10⁶ PBMC for N. (B) Correlation matrices of cellular and humoral immune variables analyzed. Only significant correlations (p<0.05) are represented with asterisks. Positive correlations appear in purple, and negative correlations appear in orange. The size and the colour gradient of the circle corresponds to the magnitude of the correlation. (C) Results of SARS-CoV-2 S1 IgG ELISA according to DPSO. Dashed line represents the established cut-off of positivity (ratio ≥1.1). (D) Neutralizing capacity of 1/500 diluted sera, tested with S protein-pseudotyped virus represented as percentage of normalized infection neutralized. (E) Correlation between neutralizing capacity and the semi-quantitative results of SARS-CoV-2 IgG ELISA (left) and anti-Spike IgG1 assessed by flow cytometry (right). Linear regressions were performed using Spearman’s rank test. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney or Wilcoxon signed rank tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. RSV: Relative Spot Volume; DP: IFN-γ/IL-2 double positive sfu.

Fig 3. Cellular and humoral immune responses in moderate natural infection, during acute and convalescent phases.

(A) FluoroSpot IFN-γ responses against SARS-CoV-2 S1, M and N proteins according to the days post-symptom onset (DPSO). Dashed lines represent the positivity cut-off established by using a non-infected control group: >25 sfu/10⁶ PBMC for S1, >21 sfu/10⁶ PBMC for M and >14 sfu/10⁶ PBMC for N. (B) Correlation matrices of cellular and humoral immune response variables analyzed. Only significant correlations (p<0.05) are represented with asterisks. (C) Results of SARS-CoV-2 S1 IgG ELISA according to DPSO. (D) Neutralizing capacity tested with S protein-pseudotyped virus represented as percentage of normalized infection neutralized. (E) Correlation between neutralizing capacity and the semi-quantitative results of SARS-CoV-2 IgG ELISA (left) and anti-Spike IgG1 assessed by flow cytometry (right). Linear regressions were performed using Spearman’s rank test. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney or Wilcoxon signed rank tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (See Fig 2 footnote for more detailed information).

Fig 4. Cellular and humoral immune responses in severe natural infection, during acute phase, measured at emergency room (ER) and a week after hospitalization (1W Hosp.).

(A) FluoroSpot IFN-γ responses against SARS-CoV-2 S1, M and N proteins according to the days post-symptom onset (DPSO). Dashed lines represent the positivity cut-off established by using a non-infected control group: >25 sfu/10⁶ PBMC for S1, >21 sfu/10⁶ PBMC for M and >14 sfu/10⁶ PBMC for N. (B) Correlation matrices of cellular and humoral immune variables analyzed. Only significant correlations (p<0.05) are represented with asterisks. (C) FluoroSpot IFN-γ responses against SARS-CoV-2 S1, M and N proteins at ER and a week after hospitalization. (D-E) Results of SARS-CoV-2 S1 IgG ELISA according to DPSO. (F) Neutralizing capacity tested with S protein-pseudotyped virus represented as percentage of normalized infection neutralized. (G) Correlation between neutralizing capacity and the semi-quantitative results of SARS-CoV-2 IgG ELISA (left) and anti-Spike IgG1 assessed by flow cytometry (right). (H-I) Comparison of S1, M and N T-cell (H) and anti-S1 IgG (I) responses between survivor and non-survivor patients (black stars). Linear regressions were performed using Spearman’s rank test. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney, Wilcoxon signed rank or Kruskal-Wallis tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (See Fig 2 footnote for more detailed information).

Fig 5. Association between dynamics of SARS-CoV-2-specific immune responses and COVID-19 severity.

(A) SARS-CoV-2-specific IFN-γ-producing T-cell responses reactive to the S1, M and N proteins in mild, moderate, and severe patients during the first (W1) and the second (W2) week post-symptom onset. (B) Relative Spot Volume (RSV) per spot of secreted IFN-γ after S1, M and N peptide pool stimulation in mild, moderate, and severe patients during acute infection. (C) Ct values obtained on real time RT-PCR for detection of the E gene upon arrival to ER in mild, moderate and severe patients. A higher Ct value corresponded to a lower viral load. (D) Correlation between S1 IFN-γ-producing T-cells and the relative viral load represented as Ct value at ER. (E) Semi-quantitative results of SARS-CoV-2 S1 IgG ELISA in the acute phase in mild, moderate, and severe patients. Dashed line represents the established cut-off of positivity (ratio ≥1.1). (F) Neutralizing capacity tested with S protein-pseudotyped virus represented as normalized infection. (G) Longitudinal data on the dynamics of cellular and humoral responses in six representative patients from the 3 severity cohorts. (H) Percentage of positive S1 cellular and humoral response during acute phase according to the days post symptom onset (DPSO) in mild, moderate and severe patients. (I) Comparison of IFN-γ responses against SARS-CoV-2 S1, M and N proteins between mild and moderate patients in convalescent phase. (J) Comparison of semi-quantitative results of SARS-CoV-2 S1 IgG between mild and moderate patients in convalescent phase. (K) Comparison of neutralizing capacity tested with S protein-pseudotyped virus represented as normalized infection. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. Linear regressions were performed using Spearman’s rank test. The significance between groups was determined using Mann Whitney, Wilcoxon signed rank or Kruskal-Wallis tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ER: Emergency Room; Conv: Convalescence; 1W Hosp.: 1 Week Hospitalization; DPSO: Days post-symptom onset.

Fig 6. Duration of SARS-CoV-2-specific cellular and humoral immunity after natural infection.

(A) S1, M and N cellular responses in recovered patients. (B) Comparison between IFN-γ and IL-2-producing T-cell responses. (C) IFN-γ-producing T-cell responses according to the months post-symptom onset (PSO). (D) Correlation matrix of cellular and humoral immune variables analyzed. Only significant correlations (p<0.05) are represented with asterisks. (E) SARS-CoV-2 S1 IgG according to the months PSO. Dashed line represents the established cut-off of positivity (ratio ≥1.1). (F) Neutralizing capacity represented as normalized infection according to the months PSO. (G) Correlation between neutralizing capacity and the semi-quantitative results of SARS-CoV-2 IgG ELISA (left) and anti-Spike IgG1 measured by flow cytometry. (H-J) S1 IFN-γ-producing T-cells (H), semi-quantitative results of SARS-CoV-2 S1 IgG (I) and neutralizing capacity (J) in recovered patients who suffered a moderate or severe acute disease. Linear regressions were performed using Spearman’s rank test. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney or Kruskal-Wallis tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. RSV: Relative Spot Volume; DP: IFN-γ/IL-2 double positive sfu.

Fig 7. Development of SARS-CoV-2-specific cellular and humoral immunity after BNT162b2 vaccination.

(A) IFN-γ-producing T-cell responses against S1, M and N proteins pre-vaccine and pre-boost. (B-D) S1 SARS-CoV-2-specific IFN-γ (B), IL-2 (C) and both cytokines (D) producing T-cells pre-vaccine, pre-boost and 15, 30 and 90 days after complete vaccination. Dashed line represents the positivity cut-off: >25 IFN-γ sfu/10⁶ PBMC. (E) Correlation matrix of adaptive immune variables analyzed. Only significant correlations (p<0.05) are represented with asterisks. (F) Frequency of CD4 Th1, Th2 and Th17 PD1+ cells pre-vaccine, pre-boost and 15 days after complete vaccination. (G) Anti-S1 IgG results pre-vaccine, pre-boost and 15, 30 and 90 days after complete vaccination. Dashed line represents the positivity cut-off (ratio ≥1.1). (H) Neutralization capacity pre-vaccine, pre-boost and 15 and 30 days after complete vaccination. (I) Correlation between neutralization and anti-S1 IgG (left) and anti-Spike IgG1 (right). (J-L) Correlation between age and S1 IFN-γ-producing T-cells (J), anti-S1 IgG (K) and neutralization (L) 15 days after full vaccination when the vaccine-elicited immune response peaked. (M) Comparison of S1 T-cell response between SARS-CoV-2 natural infection and complete vaccination. (N-O) Comparison of anti-S1 IgG (N) and neutralization (O) between SARS-CoV-2 natural infection and complete vaccination. Linear regressions were performed using Spearman’s rank test. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney, Wilcoxon signed rank or Kruskal-Wallis tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DPSO: Days post symptom onset.