key: cord-0877643-89glpbgc
authors: Kannenberg, Judith; Trawinski, Henning; Henschler, Reinhard; Buhmann, Raymund; Hönemann, Mario; Jassoy, Christian
title: Antibody course and memory B-cell response in the first year after SARS-CoV-2 infection
date: 2022-02-01
journal: J Infect Dis
DOI: 10.1093/infdis/jiac034
sha: 861fed610a8b8acb693a92a7239aed00ebcba855
doc_id: 877643
cord_uid: 89glpbgc

BACKGROUND: The possibility of repeat infections with SARS-CoV-2 raises questions regarding quality and longevity of the virus-induced immune response. METHODS: The antibody course and memory B-cell (MBC) response against SARS-CoV-2 proteins, influenza virus nucleoprotein (NP) and tetanus toxin (Ttx) were examined in adults with mild to moderate SARS-CoV-2 infection in the first year after infection. RESULTS: The concentration of SARS-CoV-2 RBD-specific antibodies was low compared with the concentration of influenza virus NP-specific antibodies. The SARS-CoV-2 RBD antibody half-life increased from 95 days in the first six months to 781 days after 9-12 months. The SARS-CoV-2 NP antibody half-life increased from 88 to 248 days. Two thirds of the subjects had SARS CoV-2-specific MBC responses 12 months after infection. SARS-CoV-2 antibody levels correlated with the MBC frequency at 12 months. CONCLUSIONS: The low concentration of SARS-CoV-2 spike protein antibodies indicates that re-exposure to the virus or vaccination are required to use the B-cell immunity to full capacity. The existence of a robust SARS CoV-2 MBC response at 12 months in most subjects and the substantially increasing antibody half-life provide evidence that the immune response is developing into long-term immunity. The early antibody reaction and the ensuing MBC response are interdependent.

Previous infection with SARS-CoV-2 protects approximately 80% of infected individuals from repeat infection for at least several months. The level of protection decreases with age and was less than 50% among individuals 65 years and older [1] . This argues for a need to further explore the magnitude and course of the virus-specific immune response. Previous studies have shown that SARS-CoV-2 infection induces a virus-specific IgG antibody response that peaks at 20-24 days after infection and subsequently declines [2] [3] [4] . It was also reported that in the first 9 months after infection the average half-lives of IgG antibodies against the viral nucleoprotein (NP) and spike (S) protein were 36-85 and 36-344 days, respectively [2, 3, [5] [6] [7] [8] [9] [10] [11] . At later time points the antibodies decayed more slowly indicating different phases of antibody decline [2, 9, 10, 12, 13] .

the MBC response increases with time from infection and reached a maximum 4-5 months after symptom onset [4, 7, 10, 12, 14, 15] . At 8-9 months after infection, SARS-CoV-2-specific MBCs were found in 69.2 to 100% of recovered subjects suggesting that the infection induces robust memory Bcell responses [4, 16] .

To examine the development of the SARS-CoV-2 antibody half-life and to test if the antibody and MBC response to SARS-CoV-2 differ from the immune response against more frequently encountered natural and vaccine antigens, we measured the concentration of antibodies against SARS-CoV-2 RBD and NP, influenza virus NP and tetanus toxin (Ttx) at different time points. We A c c e p t e d M a n u s c r i p t

Participants of the study Participants (n = 55) with SARS-CoV-2 infection and uninfected control subjects (n = 15) were recruited for the study. In infected individuals, the days post symptom onset (PSO) were counted from the first day of symptoms reported or, in the case of asymptomatic infection, the day of the first positive RT-PCR. Serum samples were collected 4 or 5 times during the first year PSO.

Heparinized blood samples were taken at 12 months PSO. Blood samples were obtained after informed consent. Sera were stored at -20°C. Heparinized blood samples were examined the same day or the day following blood drawing. The study was approved by the Ethics Commission of the Medical Faculty at the University of Leipzig (ethical vote 147/20-ek).

The concentration of antibodies against the SARS-CoV-2 receptor binding domain (RBD) in sera was determined with the Abbott SARS-CoV-2 IgG II Quant assay using the ARCHITECT i2000SR system (Abbott, Chicago, U. S. A.). This led to antibody concentrations in arbitrary units (AU)/ml. Sera below 50 AU/ml were regarded as negative. To convert the AU values into WHO binding antibody units (BAU), AU values were divided by 7 according to information from the manufacturer.

To determine the SARS-CoV-2 RBD antibody concentration in µg/ml, the human anti-SARS-CoV-2 spike S1 RBD monoclonal antibody (mAb) CR3022 (Antibodies-online.com) was examined with the Abbott SARS-CoV-2 IgG II Quant assay. The measurement showed that 520 Abbott AU were equivalent to 1 µg anti-SARS-CoV-2 RBD mAb CR3022. Therefore, the RBD antibody concentrations in AU/ml were converted into µg/ml by multiplying 1 AU with 1.92x10⁻³ µg.

A c c e p t e d M a n u s c r i p t IgG antibodies against the SARS-CoV-2 and the influenza virus nucleoproteins (NP) were measured by in-house ELISAs as previously described [17, 18] . Serial dilutions of the National Institute of Biological Standards and Controls (NIBSC) Anti-SARS-CoV-2 Antibody Diagnostic Calibrant (code 20/162) and a recombinant mAb against influenza virus NP [19] were used as concentration standards. Sera were diluted 1:100 (SARS-CoV-2 NP ELISA) or 1:5000 (influenza virus NP ELISA). 

PBMCs were isolated from heparinized blood (15 ml) by ficoll density gradient centrifugation and resuspended in RPMI-1640 medium containing 20 % fetal calf serum (FCS), penicillin, streptomycin, sodium pyruvate, non-essential amino acids, 1 µg/ml R848 (Resiquimod, Sigma-Aldrich, Merck KGaA) and 0.11 µg/ml interleukin-2 (Proleukin, Novartis AG). Cells were cultured for 5 days at 3 x10⁶ PBMC in 2 ml medium in a 24 well plate at 37°C and 5% CO 2. MBCs were examined by ELISpot using 96-well Multiscreen-IP filter plates (Millipore, Merck KGaA). The plates were washed for 15 seconds with 35% ethanol and with PBS and coated with 50 µl SARS-CoV NP-maltose binding M a n u s c r i p t protein (MBP) fusion protein (2 µg/well), SARS-CoV-2 RBD (1 µg/well [17] ), influenza virus NP-MBP fusion protein (2 µg/well) or Ttx (5 µg/well, lot 317490, GSK Vaccines). As controls, wells were coated with PBS or MBP (1 µg/well). Total numbers of IgG-secreting cells were determined with wells coated with mouse anti-human IgG mAb (clone MT91/145, Mabtech AB). The plates were incubated overnight at 4°C or for 2 hours at 37°C, washed and blocked for an hour with medium containing 20 % FCS. Stimulated cells were added to the antigen-coated wells (300,000 cells) and to anti-IgG coated wells (5,000 cells). Plates were incubated at 37°C for 20 hours.

The next day, plates were washed, alkaline phosphatase (AP)-conjugated goat anti-human IgG (no. 109-055-098, Jackson Immunoresearch Laboratories, Inc., diluted 1:5000) was added and incubated for 2 hours at 37°C. Plates were washed and NBT/BCIP substrate (AP conjugate substrate kit, Bio-Rad Laboratories, Inc.) was added for 5 minutes. Plates were washed with water, dried overnight and read with the AID EliSpot/FluoroSpot reader. Uncoated wells were used as negative control for SARS-CoV-2 RBD and Ttx. Wells coated with MBP were used as negative control for SARS-CoV-2 NP and influenza NP, because the antigens contain MBP as fusion protein [18] . Positive MBC results were defined as showing at least 10 spots per well and at least 3 times the spots in negative control wells [20] . MBCs were measured in duplicates. Mean values were calculated and the percentage of antigen-specific MBCs was calculated by the following equation:

Antibody concentrations were compared with the paired one-sided Wilcoxon signed-rank test. To calculate the antibody half-lives, the data were censored in the following way: Individuals (1) ln(y) = β 0 + β 1 T where y represents the antibody concentration, β 0 the intercept, β 1 the slope of the curve and T the days after symptom onset [21] . In the linear regression model, the mean of the individual slopes was taken as the model slope. The half-life was calculated by dividing ln(0.5) by the slopes according to equation (2).

(2) T 1/2 =ln(0.5)/β 1 95% confidence intervals (CI) of the half-lives were calculated by applying the 95% confidence intervals of the slopes in equation (2).

A c c e p t e d M a n u s c r i p t 

The subjects (n = 55) had been infected with SARS-CoV-2 between March and May 2020. RBD and NP antibodies between examinations was statistically significant during the whole study period (Suppl. Fig. 1 ). The RBD-and NP-specific antibody concentrations correlated at all time points.

Thus, participants with high antibody responses to SARS-CoV-2 RBD tended to have high antibody responses to NP as well ( Table 2 , Suppl. Fig. 2 ). (Fig. 1) . (Fig. 2) .

A c c e p t e d M a n u s c r i p t SARS-CoV-2 RBD and NP specific MBC frequencies correlated moderately with each other (rho = 0.40, p = 0.03), but did not correlate with the influenza NP or Ttx-specific MBC response (rho = -0.13 to 0.13, p = 0.49-0.99) ( Table 2 and Suppl. Fig. 4 ).

The influenza NP and tetanus toxin did no correlate ( Table 2 , Suppl. Fig. 5 ).

The goal of the study was to measure the SARS-2 RBD and NP-specific antibody M a n u s c r i p t

The influenza virus NP-specific IgG antibody concentration was more than 10 times greater than the concentration of RBD-specific IgG indicating that the SARS-CoV-2 antibody response is relatively weak compared with the antibody response against another respiratory virus. It suggests that in principle the immune system is capable to generate markedly higher antibody concentrations after appropriate stimulation. The finding also indicates that the participants have considerably more influenza NP-specific than SARS-CoV-2 RBD-specific plasma cells in the body. This finding is in line with the observation that the frequency of bone marrow residing influenza virus haemagglutinin-specific IgG-secreting plasma cells outnumbered the frequency of SARS-CoV-2 Sspecific bone marrow plasma cells in subjects who recovered from SARS-CoV-2 infection [22] .

Between Previous studies that examined the SARS-CoV-2-specific MBC response observed that the MBC frequency increased during 4-5 months after infection and the frequency of RBD-specific MBCs decreased between 6.2 and 12 months after infection [4, 7, 14, 28] . We found SARS-CoV-2-specific It has been shown that SARS-CoV-2 vaccination markedly boosts the antibody response in infected individuals [28] . This study describes the immunologic situation in which this effect occurs.

A year after infection the antibody level is comparably low, the spike-protein antibody half-life has increased to two years and most SARS-CoV-2 infected individuals have developed a robust virusspecific MBC response.

A c c e p t e d M a n u s c r i p t M a n u s c r i p t 

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We are grateful to the participants of the study. We also thank C. Schnurra, N. Reiners, A.Friedland and the team of the blood bank for support in recruiting participants and for blood drawing. We thank T. König and K. Bräutigam for technical assistance, S. Reiche and A. Aebischer for the SARS-CoV-2 RBD protein and T. Schöneberg and T. Hermsdorf for the SARS-CoV-2 NP. Many thanks also to A. Kühnapfel for statistical consulting and U. Sack for helpful discussion.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Tables A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t