SARS-CoV-2-specicific humoral immunity in convalescent patients with mild COVID-19 is supported by CD4+ T-cell help and negatively correlated with Alphacoronavirus-specific antibody titer.

This study aimed at investigating the nature of SARS-CoV-2-specific immunity in patients with mild COVID-19 and sought to identify parameters most relevant for the generation of neutralizing antibody responses in convalescent COVID-19 patients. In the majority of the examined patients a cellular as well as humoral immune response directed to SARS-CoV-2 was detected. The finding of an anti-SARS-CoV-2-reactive cellular immune response in healthy individuals suggests a pre-existing immunity to various common cold HCoVs which share close homology with SARS-CoV-2. The humoral immunity to the S protein of SARS-CoV-2 detected in convalescent COVID-19 patients correlates with the presence of SARS-CoV-2-reactive CD4+ T cells expressing Th1 cytokines. Remarkably, an inverse correlation of SARS-CoV-2 S protein-specific IgGs with HCoV-NL63 and HCoV-229E S1 protein-specific IgGs suggests that pre-existing immunity to Alphacoronaviruses might have had an inhibitory imprint on the immune response to SARS-CoV-2-infection in the examined patients with mild COVID-19.

Introduction

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) in approximately 5-15% of infected individuals. SARS-CoV-2 is a large RNA virus belonging to the Betacoronavirus genus within the family of Coronaviridae [1] infecting a wide range of hosts including man using angiotensin-converting enzyme 2 (ACE2) receptor as the dominant mechanism of cell entry [2]. While SARS-CoV and Middle East Respiratory Syndrome (MERS)-CoV have caused limited epidemics with severe pneumonia, other members of the human CoV-family, namely HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63, are endemically transmitted causing common cold with rare fatal infections of the upper and lower respiratory tract [3].

Infections with HCoV result in specific cellular and humoral immunity that can be detected several years after infection. While HCoV-specific T-cell memory remains stable and can be detected more than 11 years after SARS-CoV infection ([4, 5]), SARS-CoV- and HCoV-229E-specific antibodies titers have been shown to decline and fall below the detection limit within few years in a significant proportion of infected individuals [6], [7], [8], [9]. However, this is not surprising and a common phenomenon for primary immune responses leading to fast clearance of the pathogen.

Among members of the HCoVs many proteins, particularly the ORF1 region, are highly conserved and hence show a high homology among many HCoVs [10]. Therefore, cross-reactivity of HCoV-specific T cells with SARS-CoV-2 antigens present in individuals might contribute to the susceptibility and severity of SARS-CoV-2 infection and COVID-19 [5]. Indeed, SARS-CoV-specific T-cells responses were detected in a significant proportion of SARS-Co-2 unexposed individuals [5].

Significant reduced number of particularly CD8+ T cells found in peripheral blood of patients with moderate and severe COVID-19 cases were reported [11], [12], [13]. In contrast, the vast majority of convalescent patients or patients with mild symptoms show normal or slightly increased T cell counts [14], [15], [16]. The magnitude of T-cell lymphopenia correlates with the severity and mortality of COVID-19, thereby demonstrating the pivotal role of which T cells play for the course of the disease ([12, 13, 15]).

In convalescent COVID-19 patients the SARS-CoV-specific T-cell immunity is dominated by CD8+ T-cells directed to various structural viral proteins including M, N, S as well as ORF3, whereas CD4+ T-cell immunity was found to be mainly confined to the S protein [17]. However, T-cell specificity for SARS-CoV-2 declined and in only one-third of patients SARS-CoV-2 T-cell immunity restricted to the viral N protein was detected post recovery [18]. Another study demonstrated that SARS-CoV-2-specific CD4+ and CD8+ T-cell responses are predominantly directed to the viral S protein epitopes in COVID-19 patients with moderate to severe acute respiratory distress symptoms approximately two weeks after admission to an intensive care unit. Since durable high-affinity antibody responses depend on CD4+ T-cell help, key to the understanding of the generation of SARS-CoV-2 neutralizing antibodies and severity of the disease is the CD4+ T-cell immunity directed to the receptor binding domain (RBD) of SARS-CoV-2. The extent of humoral cross-reactivity between Alpha- and Betacoronaviruses has been analysed in natural and experimental infections studies. Antibody responses directed to common cold HCoV N proteins indicate that cross-reactivity is limited within the Alpha-HCoVs (HCov229E and HCoVNL63) and Beta-HCoVs (HCoVOK43 and HCoVHUK1), but does not occur between Alpha-HCoVs and Beta-HCoVs [19], [20], [21], [22]. Moreover, no or little humoral cross-reactivity has been observed between common cold HCoVs, SARS-CoV and MERS [23], [24], [25], [26], [27].

Material and Methods

Convalescent COVID-19 patients and healthy individuals before 2019

Heparinized whole blood samples from 15 healthy individuals (HI) collected before 2019 (age: 45.7±12.8 (mean and SD)) and 18 SARS-CoV-2 convalescent COVID-19 patients (age: 35.6±10.3 (mean and SD)) were collected between 56 and 217 days post symptoms onset by the German Red Cross - Blood Donation Service, Institute for Transfusion Medicine Dresden with the informed consent of blood donors and after consent vote of the institutional review board (EK138042014). 17 SARS-CoV-2 infected patients (5 female, 13 male) represented non-severe COVID-19 cases according to the classification of WHO. One COVID-19 patient was classified as severe COVID-19 case and hospitalized but did not require intensive care unit (ICU) care. Blood samples from 11 HI (6 female, 5 male) were collected before 2019. Blood samples form HI tested negative for SARS-CoV-2 S IgGs (2 female, 2 male) were collected before 2020.

Isolation and cryopreservation of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Hypaque as described. Briefly, heparinized whole blood were diluted with PBS/HSA (5%) (v/v) and carefully layered on Biocoll (Biochrom, Berlin, Germany) and centrifuged with 800g for 30 minutes at RT. The white layer were isolated and washed with PBS/HSA by centrifugation 300g and 5 minutes at 4°C. PBMCs were frozen in X-Vivo10 (Lonza, Basel, Switzerland) with 10% DMSO (Sigma-Aldrich, St. Louis, US-MO) and 40 mg/ml HSA (Baxter, Unterschleißheim, Germany) using controlled rate freezing containers ‘Mr. Frosty’ (Nalgene Nunc Int., Rochester, US-NY) and stored in the gas phase of a liquid nitrogen tank. Cell counts and viability were obtained using Trypan blue staining (Thermo Fisher Scientific, Waltham, US-MA) and a TC20TM automated cell counter (Biorad).

Stimulation of PBMCs with SARS-CoV-2 and CMV pp65 peptide mixes

To detect virus-peptide-reactive CD3+ T cells approximately 1.5 × 106 PBMCs were stimulated with SARS-Cov-2 M, N, S and S1 (Miltenyi Biotec, Bergisch Gladbach, Germany) or CMV pp65 peptide mixes (JPT Peptide Technologies, Berlin, Germany) consisting of 15-mers overlapping by 11 amino acids at a concentration of 1µM/per single peptide in RPMI1640 with 10 mg/ml HSA in a 96-well round bottom plate (Greiner Bio-one, Kremsmuenster, Austria) at 37°C in a humidified atmosphere. In addition, αCD28 (clone: L293) (BD Biosciences) at a final concentration of 1.3µg/ml and a total volume of 150µl/well was added as a co-stimulatory signal to each well. As negative control PBMCs were stimulated with αCD28 alone. To enable detection of intracellular cytokines 1 hour after stimulation with peptide mixes and aCD28 mAb, GolgiPlugTM (BD Biosciences) was added to every well followed by an additional 4 hours of incubation. Cells were then washed with PBS/HSA (5 mg/ml) and stained with Ethidium monoazide (EMA) (Thermo Fisher Scientific) prior to fixation with Cytofix/Cytoperm solution (BD Biosciences). Subsequently, monoclonal antibody staining was performed with αCD3 BV421 (clone: UCHT1), αCD4 APC-Vio770 (clone: M-T321) (Miltenyi Biotec), αCD8 V500 (clone: SK1), αIFN-γ FITC (clone: 25723.11), αTNF-α PE-Cy7 (clone: Mab11) (all BD Biosciences) and αIL-2 PE (clone: MQ1-17H12) (eBioscience, San Diego, US). Gates were set according to FMO controls. T-cell assays, e.g. ICS, presented in this work were performed compliant to MIATA guidelines.

Flow cytometric analysis

For flow cytometric analysis were performed using a FACS Canto II equipped with three lasers (blue 488 nm, red 633 nm and violet 405 nm) and Diva-Software V6.1.3 (both BD Bioscience) and adhered to the guidelines for the use of flow cytometry and cell sorting in immunological studies [28]. PMT voltages were adjusted to yield optimal signal to noise ratios. Compensation was applied for each fluorochrome. Gating strategies and a representative data set are shown in supplementary figure 1. Data analyses was performed using FlowJo software V9.3.2 (FlowJo LLC, Ashland, US-OR).

Quantification and statistical analysis

Statistical data were calculated using GraphPad Prism software V6.02 (GraphPad Software Inc., La Jolla, US-CA). For the identification and determination of the frequency of intracellularly cytokine producing, antigen-reactive T cells the gating strategy as displayed in Supplementary Figure 1 was applied. In order to evaluate the presence of SARS-CoV-2-specific T-cell immunity a Stimulation Index (SI) was calculated as following: percentage of intracellular cytokine positive T cells following stimulation with SARS-CoV-2 peptide mixes (n=1) divided by the average (arithmetric) mean value of the percentage of intracellular cytokine positive T cells (from 3 replicative measurements, n=3) following incubation with αCD28 alone plus its SD. T-cell responses with a SI were rated positive. The frequency of SARS-CoV-2- and CMV pp65-specific T cells immunity given in Figures and in Tables was determined by subtracting the average arithmetic mean frequency of intracellular cytokine positive T cells detected following three independent stimulation with aCD28 alone (n=3) from the frequency of intracellular cytokine positive T cells detected following stimulation with peptide mixes. Statistical significance (p) of antigen-reactive T cells frequencies between various groups was calculated using Student´s unpaired t-test.

Virus neutralization assay

Plaque reduction neutralization test (PRNT) were performed as described before [29]. Briefly, 4 × 105 cells/ml VeroE6 cells were seeded in 24-well plates 1 day prior testing. Patient plasma was heat-inactivated at 56°C for 30 minutes, diluted with OptiPro medium (Fisher scientific, Schwerte, Germany) starting from a titer of 1:20 to 1:640 and incubated with a solution containing 100 plaque forming units of SARS-CoV-2 at 37°C for 1h. The virus containing plasma were then added in duplicates to the wells of 24-well plates and incubated at 37°C. After 1 hour the supernatant was discarded and the cells were washed once with PBS and supplemented with 1.2% Avicel solution in DMEM (Merck, Darmstadt, Germany). After 3 days incubation at 37°C the supernatants were removed and the cells were fixed with PBS containing 6% formaldehyde and stained with crystal violet as described [30].

Determination of SARS-CoV-2-S protein-specific IgG

SARS CoV-2 IgG titre was determined using a CE-marked anti-SARS-CoV-2 IgG semiquantitative ELISA (Euroimmun, Lübeck, Germany) according to the manufacturer´s instructions. Results are given as the ratio of the optical density of the patient sample divided by arbitrary unit ratio of the provided control sample antibody. Antibody ratios (AbR) of ≥0.8 were considered positive.

Detection of SARS-CoV-2-, SARS-CoV-1-, MERS-CoV- and HCoV-specific IgG using multiplex antibody bead array

In order to extend the characterization of the humoral response to additional SARS-CoV-2 antigens including the RBD, nucleocapsid (N), S1/S2 domain and to the S1 domain of various relevant HCoVs (HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63) the serum of reconvalescent COVID-19 patients was further assessed using the SARS-CoV-2 specific multiplex antibody detection array (MABA) (Labscreen COVID plus) (One lambda, West Hill, CA, USA) and HLA Fusion software (Luminex, Austin, TX, USA)). Briefly, prior detection of SARS-CoV-2-specific IgGs 1µl EDTA (0.2M) was added to 20µl of diluted (1:10) human serum. 20µl diluted human serum was incubated with 5µl Labscreen COVID plus multiplex beads for 30 min at RT in the dark. Following washing three times with PBS/HSA buffer multiplex beads were resuspended with 100µl human IgG-PE and incubated for additional 30 min at RT in the dark. After washing two times with PBS/HSA the multiplex beads were resuspended in 80µl PBS and analysed using Luminex xMAPbased assay and a LABScan3D (Luminex, Austin, TX, USA) and Microsoft Excel. Cutoff values for each recombinant protein were calculated and given as stated by the manufacturer using the mean MFI + 3xSD of 96 COVID-19 negative samples collected before 2019 [31].

Results

SARS-CoV-2 M, N and S/S1 protein-reactive T-cell immunity

SARS-CoV-2-infections have been shown to trigger Th1 responses with specificity to the structural immunodominant viral M, N and S protein. The S1 subdomain comprises the RBD within the S protein. Therefore, the SARS-CoV-2-specific T-cell immunity was examined for the presence of T cells with reactivity to the M, N, S and S1 protein in convalescent COVID-19 patients by ICS for IL-2, IFN-γ and TNF-α following stimulation with SARS-CoV-2-peptide mixes.

In total, PBMCs of 18 convalescent COVID-19 patients were analysed for SARS-CoV-2-reactive T-cell immunity. To identify patients with SARS-CoV-2-reactive T-cell responses SI were calculated and patient with a SI3 were rated to manifest SARS-CoV-2-reactive T-cell immunity (Supplementary Figure 2). In 17 COVID-19 patients (94%) SARS-CoV-2- reactive IL-2 and/or IFN-γ producing CD4+ or CD8+ T cells were detected. CD4+ SARS-CoV-2- reactive immunity was found in 16 (89%) COVID-19 patients which was directed to the SARS-CoV-2 M (n=11) (61%), N (n=10) (56%), S (n=13) (72%) or S1 protein (n=12) (67%). In contrast, only 10 (56%) of these COVID-19 patients demonstrated SARS-CoV-2- reactive IL-2 or IFN-γ producing CD8+ T cells with a specificity for the SARS-CoV-2 M (n=7) (39%), N (n=7) (39%), S (n=7) (39%) and S1 protein (n=4) (22%).

Also in the majority of the tested HI SARS-CoV-2-reactive T-cell immunity was detectable. In 12 out of 15 HI (80%) SARS-CoV-2-reactive CD4+ T-cell immunity and in 9 (60%) CD8+ T-cell immunity reactive for at least one of the tested SARS-CoV-2 peptide mix was detected. Similar to the COVID-19 patients CD4+ T cells reactive for all tested SARS-CoV-2 peptide mixes without prevalence could be detected (M (n=6) (40%), N (n=5) (33%), S (n=7) (47%) or S1 protein (n=3) (20%). In contrast, CD8+ T-cell immunity showed predominant reactivity for SARS-CoV-2 N and S/S1 proteins M (n=3) (20%), N (n=5) (33%), S (n=5) (33%) or S1 protein (n=5) (33%).

Remarkably, the determination of the SI of SARS-CoV-2-reactive CD4+ and CD8+ T cells was hampered by an abnormal high background of spontaneously TNF-α producing T cells in several COVID-19 patients. This becomes evident in discrepant SI found for CMV pp65- reactive T cell population producing various cytokines (Supplementary Figure 2). While in COVID-19 patients displaying a high frequency of spontaneously producing TNF-α a negative SI for TNFα+ T cells reactive for CMV pp65 was calculated, positive SI (3) and significant frequencies of IFN-γ and IL-2 producing T cells following stimulation with CMV pp65 peptide mix were determined. In general, intracellular TNF-α expression in T cells was associated with IFN-γ co-expression.

With regard to frequency the SARS-CoV-2-reactive T-cell immunity in the analysed COVID-19 convalescent patients is clearly dominated by CD4+ T cells reactive for the M protein. Median frequencies of 0.012% (ranging: -0.008%-0.410%) and 0.002% (ranging: -0.108%-0.395%) SARS-CoV-2 M protein-reactive IFN-γ+ and TNF-α+ CD4+ T cells in convalescent COVID-19 patients were observed (Figure 1 b and c, Table 1 ). However, compared to the median frequencies of SARS-CoV-2 M protein-reactive IFN-γ+ CD4+ T cells and TNF-α+CD4+ T cells detected in PBMCs from HI the median frequencies were not increased (Figure 1b, Table 2 ). Also, median frequencies of N, S and S1 protein-reactive of IFN-γ+ and TNF-α+CD4+ T cells detected in COVID-19 patients were not increased compared to the frequencies detected in HI (Figure 1b and c, Table 1 and 2).

Figure 1

Frequency of SARS-CoV-2 M, N, S, S1 protein-reactive T cells detected in PBMCs of convalescent COVID-19 patients and healthy individuals.

Frequency of IFN-γ, IL-2 and TNF-α expressing CD4+ or CD8+ T cells detected after stimulation with respective SARS-CoV-2 M, N and S/S peptide mixes among T cells from convalescent COVID-19 patients and HI. Each closed black circles represent a single measurement of one convalescent COVID-19 patients. Each grey triangle represent a single measurement of one HIs. Symbols and bars represent median and range. Statistically significant differences (p<0.05) between groups of antigen-reactive T cells are indicated with horizontal bars and *. Highly statistical differences (p<0.01) are indicated with horizontal bars and **.

Table 1

Median frequency of SARS-CoV-2 Protein-specific CD3+ T cells subpopulation in COVID-19 patients

Table 1. Frequency of SARS-CoV-2 protein-reactive CD4+ and CD8+ T cells in convalescent COVID-19 patients.

Antigen		M	N	S	S1	CMV pp65
CD4+	IL-2	0.013	0.006	0.010	0.009	0.014
	IFN-γ	0.012	0.006	0.002	0.007	0.008
	TNF-α	0.002	0.005	0.022	-0.006	0.013
CD8+	IL-2	0.004	0.002	0.006	0.000	0.013
	IFN-γ	−0.003	0.014	0.010	0.003	0.008
	TNF-α	0.004	-0.007	-0.008	-0.003	0.133

Median frequencies of SARS-CoV-2 M, N, S/S1 protein and CMV pp65-reactive IFN-γ+/IL-2+/TNF-α+CD4+ T cells and CD8+ T cells detected in PBMCs of HI.

Table 2

Median frequency of SARS-CoV-2 Protein-specific CD3+ T cells subpopulation in HI (peripheral blood collected prior 2020)

Table 2. Frequency of SARS-CoV-2 protein-reactive CD4+ and CD8+ T cells in HIs.

Antigen		M	N	S	S1	CMV pp65
CD4+	IL-2	-0.001	-0.002	0,002	0,004	0.005
	IFN-γ	0.012	0.004	0.002	0.007	0.083
	TNF-α	0.008	-0.001	0.007	-0.001	0.040
CD8+	IL-2	-0.003	-0.003	0.001	0.003	0.004
	IFN-γ	−0.003	-0.003	-0.01	-0.006	0.482
	TNF-α	-0.008	-0.023	-0.012	-0.006	0.286

Statistically significant differences between frequencies of SARS-CoV-2 M, N and S protein- reactive IL-2+CD4+ T cells detected in COVID-19 patients and HI were found. The frequencies of SARS-CoV-2 M protein-reactive IL-2+CD4+ T cells detected in convalescent COVID-19 patients (median: 0.013%, range: -0.023%-0.108%) were significantly higher (p=0.036) compared to the frequency found in HI (median: -0.001%, range: -0.006%-0.040%) (Figure 1a, Table 1 and 2). In addition, the frequency of SARS-CoV-2 N, S and S1 protein-reactive IL-2+CD4+ T cells detected in convalescent COVID-19 patients (median: 0.006%, range: -0.014%-0.074%) and (median: 0.010%, range: -0.014%-0.046%), (median: 0.009%, range: -0.023%-0.056%) (Table 1), respectively were significantly increased (p=0.045 and p=0.017, respectively) compared to HI demonstrating low frequencies of SARS-CoV-2 N, S and S1 protein-reactive IL-2+CD4+ T cells (median: -0.002%, range: -0.010%-0.017%), (median: 0.002%, range: -0.008%-0.037%) and (median: 0.004%, range: -0.007%-0.022%) (Figure 1a, Table 2).

In comparison to the frequencies of SARS-CoV-2-reactive CD4+ T cells the detected frequencies of SARS-CoV-2-reactive CD8+ T cells are rather low (Figures 1d-f). Moreover, in contrast to CD4+ T cells, SARS-CoV-2-reactive CD8+ T-cell immunity is dominated by IFN-γ+CD8+ T cells reactive for the N protein (median: 0.014%, range: -0.022%-0.134%) S and S1 protein (median: 0.010%, range: -0.075%-0.065%) (median: 0.003%, range: -0.054%-0.065%) (Figure 1e, Table 1). Frequencies of IFN-γ+CD8+ T cells were statistically significant higher compared to those detected in HI (N: median: -0.003%, range: -0.078%-0.353%) (p=0,011), (S: median: -0.010%, range: -0.070%-0.009%) (p=0.003), and (S1 protein: median: -0.010%, range: -0.070%-0.009%) (p=0.034) (Figure 1e, Table 2), respectively.

Considerable relevant frequencies of SARS-CoV-2-reactive CD8+ T cells producing IL-2 were limited to convalescent COVID-19 patients (Figure 1d). The median frequencies for SARS-CoV-2 M and S protein-reactive were 0.004% (range: -0.004-0.031) and 0.006% (range: -0.004-0.04) (Table 1).

CMV pp65-reactive T-cell immunity was detected with IL-2+ CD4+/IFN-γ+CD4+ and IL-2+ CD8+/IFN-γ+ CD8+ T cells in eleven (61%) convalescent COVID-19 patients and in 11 out of 16 analysed (69%) HI (Figure 1d and e). In five COVID-19 patients CMV pp65-reactive T cell immunity characterized by TNF-α production was disguised by high background of spontaneously TNF-α-producing T cells (Figure 1f).

Significant increase of SARS-CoV-2 N and S protein-reactive T-cell immunity in convalescent patients up to 256 days after onset of disease

SARS-CoV-2-specific T cell immunity revealed a moderate increase and correlation of IFN-γ+CD4+ T-cells reactive for the SARSCoV-2 N- and S-protein (r2=0.125, p=0.0044 and r2=0.192, p=0.012) (Figure 2 a and b) progressing with time following infection indicating generation and maintenance of a robust SARS-CoV-2-specific cellular immunity for these SARS-CoV-2 antigen in convalescent patients up to 256 days after diagnosis. No significant correlation was found between IFN-γ+CD4+ T cell immunity reactive for the SARSCoV-2 M- and S1-protein (data no shown).

Figure 2

Generation and maintenance of SARS-CoV-2-reactive IFN-γ+CD4+ T cells in con-valescent patients up to 256 days after onset of disease.

Frequency of SARS-CoV-2-N or S peptide mix-reactive IFN-γ+CD4+ T cells in convalescent patients up to 256 days after onset of disease, respectively (Figure 2a and b). Regression lines, coefficient of variations (r2) and statistical significance (p-value) are depicted in the Figures. Each symbol represents a single measurement.

Determination of humoral immunity towards SARS-CoV-2 and HCoVs

Anti-SARS-CoV-2-specific antibody responses were analysed with three different methods. Using the anti-SARS-CoV-2 S IgG ELISA relative amounts of serum IgG specific for the SARS-CoV-2 S protein, which comprises the RBD, in convalescent COVID-19 patients were determined. In addition, SARS-CoV-2 neutralizing antibodies were measured with the PRNT. MABA was used to detect the HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63 and SARS-CoV-2 S1 protein-specific IgGs including the RBD, N and S1/S2 protein of SARS-CoV-2. In order to evaluate the interrelation of the different assays used for the detection of humoral immune responses to SARS-CoV-2 and HCoVs the results from respective assays were analysed and compared. A significant correlation was found between the concentration of anti-SARS-CoV-2 S protein-specific IgGs determined by ELISA and anti-S1 protein-specific IgGs (r2=0.696, p=0.0027) (Supplementary Figure 3a) or anti-SARS-CoV-2 S (r2=0.739, p=0.0014) (Supplementary Figure 3b), detected by MABA with a more stringent correlation between IgGs specifically binding to the S protein.

Additionally, significant correlations were found between neutralizing SARS-CoV-2 antibodies and SARS-CoV-2 S protein-specific IgGs, detected by ELISA (r2=0.456, p=0.0081) (Supplementary Figure 4a) and anti-SARS-CoV-S1 protein-specific IgGs, detected by MABA (r2=0.290, p=0.0026) (Supplementary Figure 4b).

Interestingly, an inverse correlation was found between anti-HCoV-NL63 S1 and anti-HCoV-229E S1 protein-specific IgGs, detected by MABA, and anti-SARS-CoV S protein-specific IgGs, detected by ELISA (r2=0.415, p=0.044 or r2=0.625, p=0.007), respectively (Figure 3 a and b). Furthermore, an inverse correlation was detected between anti-HCoV-NL63 S1 protein-specific IgGs and neutralizing antibodies (r2=0.236, p=0.041) (Figure 3c).

Figure 3

Correlation of anti-HCoV-NL63 S1/anti-HCoV-229E S1 protein-specific IgGs levels with anti-SARS-CoV-2 S/S1-specific IgGs levels and SARS-CoV-2 neutralizing antibody titer.

MFI values of anti-SARS-CoV-2 S1-specific IgGs levels determined by MABA were compared with MFI values of anti-HCoV-NL63 S1-protein-specific IgGs (Figure 2a) or anti-HCoV-2-229E S1-protein-specific IgGs (Figure 2b) also determined by MABA in sera of convalescent COVID-19 patients. The titer of SARS-CoV-2 neutralizing antibodies is correlated to MFI values of anti-HCoV-NL63 S1-protein-specific IgGs (Figure 2c). Regression lines, coefficient of variations (r2) and statistical significance (p-value) are depicted in the Figures. Each symbol represents a single measurement.

Correlation of cellular and humoral immunity towards SARS-CoV-2 and HCoVs

A strong correlation was found between the SARS CoV-2 S neutralizing antibody titers and the frequency of S1 protein-reactive IFN-γ+CD4+ T cells (r2=0.653, p=0.002) (Figure 4 ), respectively. In contrast, no relevant correlations were revealed between SARS-CoV-2 S protein- reactive IgG and M, N or S1protein-reactive IFN-γ+CD4+ T cells or S protein-reactive IL-2+CD4+ T cells (data not shown).

Figure 4

Correlation of SARS-CoV-2 neutralizing antibody titer with the frequency of anti-SARS-CoV-2 S1 protein-reactive INF-γ+CD4+ T cells detected in convalescent COVID-19 patients.

Titer of SARS-CoV-2 neutralizing antibodies were compared with the frequency (Figure 3) of anti-SARS-CoV-2 S1 protein-reactive INF-γ+ CD4+ T cells detected in convalescent COVID-19 patients. Regression lines, coefficient of variations (r2) and statistical significance (p-value) are depicted in the Figure. Each symbol represents a single measurement.

Discussion

In our study we investigated the nature of SARS-CoV-2-specific immunity in patients with mild COVID-19 and sought to identify parameters most relevant for the generation and maintenance of neutralizing antibody responses in convalescent COVID-19 patients. The cohort of convalescent COVID-19 patients analysed and presented here comprises 18 relative young individuals with PCR-confirmed SARS-CoV-2 infection. These COVID-19 patients except one showed mild disease symptoms without the requirement of hospitalization. After recovery from SARS-CoV-2 infection they were selected as potential donors for convalescent plasma containing anti-SARS-CoV2 neutralizing antibodies according to the amount of anti-SARS-CoV-2-specific IgGs detected in their serum.

In order to investigate the correlation of the humoral response against SARS-CoV-2 and various `common cold´ HCoVs we extended our analysis using MABA. The SARS-CoV-2-reactive T-cell immunity in the examined cohort of convalescent donors was investigated using ICS following provocation with SARS-CoV-2 M, N, S and S1 protein-specific peptide mixes and compared to HI who donated blood before the beginning of the pandemic.

Measurements of the SARS-CoV-2-specific humoral immunity using various assays gave comparable results with statistically significant correlations. Highly significant correlation between the levels of SARS-CoV-2 S-protein-specific IgGs detected by ELISA and MABA showed the good comparability of both methods. Slightly reduced correlation coefficient of SARS-CoV-2 S protein-specific IgG and SARS-CoV-2 S1 protein-specific confirmed presence of SARS-CoV-2 S1 protein-specific IgGs in convalescent COVID-19 patients. However, a significant amount of SARS-CoV-2 S protein-specific IgGs are directed to the S1 protein. Importantly, the neutralizing capacity of detected SARS-CoV-2 S/S1 protein-specific IgG was demonstrated by significant correlation of the results yielded by the PRNT. The comparison of the SARS-CoV-2 S protein-specific IgG values also indicates a certain degree of variation in the detection of SARS-CoV-2-specific epitopes, particularly with respect to the RBD.

While strong antibody responses to MERS and SARS-CoV-2 coincide with the clinical course of the disease, magnitude and character of the T-cell immunity had been reported being less affected by the severity of the disease and SARS-CoV-2-specific T-cell immunity present in the majority of COVID-19 patients ([7, 18, 32, 33]). Since neutralizing, durable antibody responses and affinity-matured B cell memory depend on CD4+ T-cell help [34], key to the understanding of humoral immune response directed towards SARS-CoV-2 is the elucidation of T-cell immunity and epitopes recognized by CD4+ T cells.

Several publications described SARS-CoV-2 epitopes recognized by CD4+ and CD8+ T cells after infection and reported that the majority of CD4+ T-cell mediated SARS-CoV-2-specific immunity is directed towards structural proteins with the highest frequencies towards S/S1, M and N proteins ([33, 35, 36]). In our study we detected comparatively high frequencies M and N protein-specific and low frequencies of S/S1 protein-reactive CD4+ T cells possibly due to the examined patient cohort exhibiting mild COVID-19. However, following up the SARS-CoV-2 reactive T-cell immunity we were able to detect an increase and statistically significant correlation of IFN-γ+CD4+ T-cell immunity reactive for SARS-CoV-2 N- and S-protein in the analysed COVID-19 patients over time indicating generation and maintenance of SARS-CoV-2-specific cellular immunity in convalescent patients for more than 8 months (256 days) after onset of disease. This has been previously shown by Mazzoni et al., ([37, 38]). Of note, CD4+ T-cell immunity following infection with common cold HCoVs had been reported being predominantly directed towards the S protein [17], whereas convalescent MERS patients show a more diverse T-cell immunity directed towards M, N, and S protein [39].

In comparison to CD4+ T cells, SARS-CoV-2-specific CD8+ T-cell immunity has been described to be preferentially directed to S and N protein following infections with SARS-CoV or SARS-CoV-2. A striking predominance of S protein-reactive CD8+ T-cell immunity was found in patients infected with SARS-CoV. There, S protein-reactive CD8+ T cells amounted to approximately 50% of total detected SARS-CoV-reactive T cells whereas N protein-specific CD8+ T cells accounted for 36% [17] SARS-CoV-reactive CD8+ T cells. A similar finding was reported by Thieme et al. analysing T-cell immunity in COVID-19 patients [33]. A predominant S and N protein-reactive CD8+ T-cell immunity following SARS-CoV-2 infection was described for COVID-19, which is in good agreement with our findings presented here. However, studies analysing the T-cell immunity in COVID-19 patients reported that CD8+ T-cell immunity were predominantly directed to the N protein or M, N, and S protein without precedence ([33, 40]).

With respect to the magnitude of SARS-CoV-2-reactive T-cell immunity we detected markedly higher CD4+ T-cell responses in terms of frequency compared to CD8+ mediated T-cell responses. Also, this finding is in good agreement with various other studies ([33, 35, 41]).

Furthermore, we could confirm the presence of SARS-CoV-2-reactive T-cell immunity in the vast majority of tested convalescent COVID-19 patients. This is in line with the study of Grifoni et al. reporting on the presence of SARS-CoV-2-reactive CD4+ T cells in 100% and CD8+ T cells in 70% of tested COVID-19 patients [35]. Other studies stated lower proportions for various reasons [33]. However, the majority of the studies demonstrated that the predominant portion of tested COVID-19 patients revealed robust SARS-CoV-2-reactive T-cell immunity [37], even in absence of SARS-CoV-2-specific antibody [42]. Observed discrepancies between these findings can be possibly attributed to relative small sample numbers, different T-cell stimulation and staining methods and variable cell activation marker (CD137, CD154, CD69 or intracellular cytokines) used for the identification of SARS-CoV-2-reactive T cells.

While SARS-CoV and MERS-reactive T-cell immunity was confirmed to be persistent for many years following infection humoral immunity waned within months and virus-specific antibodies are not detectable in the majority of the virus-infected individuals after 2-3 years. For the induction of high affinity antibody responses and persistent humoral immunity CD4+ T-cell help is required. Therefore, evidence for correlation of SARS-CoV-2-reactive CD4+ T-cell immunity and SARS-CoV-2-specific humoral immunity can be taken as a strong indicator for the induction of a robust and durable SARS-CoV-2-specific humoral immunity. Our finding, that there is a significant correlation between SARS-CoV-2 neutralizing antibody titers and S/S1 protein-reactive IFN-γ+CD4+ T cells, provides further evidence for the induction of a robust humoral immunity directed to the S/S1 protein of SARS-CoV-2 in convalescent COVID-19 patients as described before ([37, 38, 40]).

Remarkably, the presence anti-HCoV-NL63 and anti-HCoV-229E S1 protein-specific IgGs is inversely correlated with the induction of humoral immunity directed to the SARS-CoV-2 S protein suggesting a potential suppressive impact of preexisting anti-HCoV-NL63 and anti-HCoV-229E S1 protein-specific IgG on the induction of humoral immunity specific for SARS-CoV-2. A negative impact of pre-existing humoral immunity specific for common cold HCoVs on the induction of SARS-CoV-2 specific antibody responses has been recently published. Aydillo et al., reported that antibodies specific for the conserved region of the common cold HCoVs spike protein were boosted in COVID-19 patients which in turn had a negative impact on the induction of antibodies against SARS-CoV-2 S and N proteins [43].

However, no such immunological imprint on the induction of SARS-CoV-2-specific antibodies was observed for HCoV-229E. HCoV-NL63 was not analysed in this analysis. In another report Lin et. al described that pre-existing humoral immunity to HCoVs S protein SARS-CoV-2 impedes the generation of SARS-CoV-2 neutralizing antibodies in mice [44].

The inhibitory effect of existing anti-HCoV-NL63 and anti-HCoV-229E S1 protein-specific IgGs may result from the suppression of activation of naive B cells and their subsequent proliferation and differentiation to plasma cells when a simultaneous binding of the antigen in the immune complex to the B cell receptor and to the inhibitory Fc-γRIIB occurs [45]. A well-known example is the failure of seroconversion of babies when vaccinated against measles within 6 months after birth as a result of the acquisition of MV-specific antibodies via transplacental transfer of maternal IgGs. Since maternal IgGs have limited half-live MV vaccinations later are much more efficient [46]. Likewise, investigations in cynomolgus macaques showed that even low pre-existing antibody titers can inhibit the generation of antibodies following vaccination with MV or recombinant vaccinia virus vector expressing measles antigens [47]. Furthermore, cross-linking of Fc-γRIIB induces apoptosis in bone marrow plasma cells [48] which consequently results in decreasing antibody levels. In addition, engagement of Fc-γRIIB by IgG immune complexes led to the failure of DC maturation resulting in inefficient CD4+ T-cells support required for B-cell activation and plasma cell differentiation [49].

Of note, the levels of S1 protein-specific IgGs of HCoV-OC43 and HCoV-HKU1 sharing a higher homology with SARS-CoV-2 [50] than HCoV-229E, were found to be clearly lower compared to S1 protein-specific IgG levels of HCoV-229E. The higher concentration of protein-specific IgG levels of HCoV-229E might have contributed to the contradicting finding concerning the inhibitory effect of pre-existing antibodies specific for the S protein of common cold Alpha- and Betacoronavirus.

Whether cross-reactive HCoVs-specific immunity is beneficial or detrimental with regard to the susceptibility to SARS-CoV-2 infection or severity of COVID-19 still remains elusive. Recent findings indicate that pre-existing immunity to seasonal coronaviruses may increase the susceptibility to SARS-CoV-2 [51]. In the context of convalescent plasma transfusion with its proven clinical benefit for critically ill COVID-19 patients ([52, 53]) the presence of anti-HCoV-NL63 and anti-HCoV-229E IgG might have an adverse impact on the therapy outcome and should therefore be considered.

Limitation of study

Using peptide mixes covering M, N, S and its subdomain S1 protein bears the risk that a large portion of potential SARS-CoV-2-reactive T cells are missed by the analysis. Low frequency of detected SARS-CoV-2-reactive T cells accompanied by an unusual high background staining – particularly for TNF-α expressing CD8+ T cells – hampered the analysis of SARS-CoV-2-reactive cellular immunity in many cases. Therefore, we confined our study to the analysis of IL-2 and IFN-γ. Although previous studies confirmed the predominant Th1 nature of the SARS-CoV-2 T-cell immunity ([36, 41, 54]), particularly directed to SARS-CoV-2 S protein in patients with mildly symptomatic COVID-19 after three months of infection [55] - the detected T-cell reactivity may not represents all facets of SARS-CoV-2-specific T-cell immunity in convalescent COVID-19 patients.

Testing of the humoral immune response specific for SARS-CoV-2 M protein was not performed due to the limitations of available test kits. Considering the predominance of SARS-CoV-2 M protein- reactive CD4+ T cells found in COVID-19 patients our study might not fully assess the role of M protein-specific humoral immunity in the context of SARS-CoV-2 infection.

In addition, the small cohort of convalescent patients of relative young age exhibiting mild to moderate COVID-19 may have contributed to biasing the detected humoral and cellular SARS-CoV-2-specific immunity. Further studies should address the issue how pre-existing, cross-reactive immunity from prior infections with common cold HCoVs have an impact on SARS-CoV-2 infection and immunity.

Conclusion

In the majority of the examined mild COVID-19 patients and HIs recruited before 2019 an immune response directed to various SARS-CoV-2 structural proteins was detected. The finding of an anti-SARS-CoV-2-reactive cellular immune response in HI suggests a pre-existing immunity to common cold HCoVs which share homology with SARS-CoV-2. The humoral immunity to the S protein of SARS-CoV-2 detected in convalescent COVID-19 patients positively correlates with the presence of SARS-CoV-2-reactive CD4+ T cells expressing Th1 cytokines indicating the generation of a robust cellular and humoral SARS-CoV-2-directed immunity. The inverse correlation of SARS-CoV-2 S protein-specific IgGs with HCoV-NL63 and HCoV-229E S1 protein-specific IgGs suggest that pre-existing humoral immunity to common cold HCoVs, particularly Alphacoronaviruses, may have had an inhibitory imprint on the humoral immune response to SARS-CoV-2 infection detected in patients with mild COVID-19.

Declaration of Competing Interest

All authors declare no conflict of interest.