key: cord-0335116-9p2pzsx0
authors: Marcotte, Harold; Piralla, Antonio; Zuo, Fanglei; Du, Likun; Cassaniti, Irene; Wan, Hui; Kumagai-Braesh, Makiko; Andréll, Juni; Percivalle, Elena; Sammartino, Josè Camilla; Wang, Yating; Vlachiotis, Stelios; Attevall, Janine; Bergami, Federica; Ferrari, Alessandro; Colaneri, Marta; Vecchia, Marco; Sambo, Margherita; Zuccaro, Valentina; Asperges, Erika; Bruno, Raffaele; Oggionni, Tiberio; Meloni, Federica; Abolhassanni, Hassan; Bertoglio, Federico; Schubert, Maren; Calzolai, Luigi; Varani, Luca; Hust, Michael; Xue, Yintong; Hammarström, Lennart; Baldanti, Fausto; Pan-Hammarström, Qiang
title: Immunity to SARS-CoV-2 up to 15 months after infection
date: 2021-10-11
journal: bioRxiv
DOI: 10.1101/2021.10.08.463699
sha: 7cd43dc9a54793b72118cca2b426758f83df55b5
doc_id: 335116
cord_uid: 9p2pzsx0

Background Information concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset. Methods The levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison. Findings Following a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months. Conclusion The data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies. Funding EU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.

Information concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset.

The levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison.

Following a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months.

Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16-and 8fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months.

The data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-

Coronavirus disease 2019 , caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly resulted in a pandemic constituting a global health emergency. The COVID-19 pathological process exhibits a wide spectrum of clinical manifestations, ranging from asymptomatic to mild, moderate, severe and critical disease. The genome of SARS-CoV-2 encodes four major structural proteins that occur in all coronavirus species: spike protein (S), nucleoprotein (N), membrane protein (M), and envelope protein (E) 1 . The S protein binds to the host receptor (Angiotensin Converting Enzyme 2 [ACE2]) through the receptor-binding domain (RBD) in the S1 subunit, followed by the S2 subunit-mediated cell membrane fusion 2 .

The adaptive immune response is likely to be critical for the development of protective immunity to SARS-CoV-2 including viral clearance and the persistence of antiviral immunity 25 .

Generation of neutralizing antibodies that specifically target the receptor-binding domain (RBD) of the S protein is considered to be essential in controlling SARS-CoV-2 infection 3, 4 . A robust adaptive immune response with presence of RBD and S-specific neutralizing antibodies, memory B cells and T cell response have been found in patients who have recovered from infection [5] [6] [7] . Although circulating antibodies derived from plasma cells wane over time, long-lived immunological memory can persist in expanded clones of memory B cells 7 .

Since December 2020, variants of concern (VOCs), which were initially named based on the country where they were first identified, have been characterized, including Alpha (B.1.1.7, UK), Beta (B.1.351, South Africa), Gamma (P.1, Brazil) and Delta (B.1.617.2, India). Mutations in the S1 subunit may lead to changes in the structure of the S protein and RBD 8 and result in higher binding of the virus to the receptor 9 , increased risk of transmission and severity of illness 10 , as well as a reduction in neutralization susceptibility by antibodies [11] [12] [13] . The Delta variant is associated with an estimated 60% higher risk of household transmission than the Alpha variant and is becoming dominant worldwide 14 .

We have previously reported the longevity of the SARS-CoV-2 adaptive immune response (up to 6-8 months) in cohorts of Swedish and Italian patients infected with the SARS-CoV-2 G614 strain 6 . The antibody and neutralizing titers were sustained at a relatively high level for at least 6 months after the onset of symptoms while specific memory B and T cells were maintained for at least 6-8 months. In this study, the adaptive immune response in convalescent patients from the same cohorts was followed up to 15 months. In addition, the specific antibody levels and neutralizing antibody titers were tested against VOCs.

For the entire 15 months follow-up, a total of 188 serum or plasma samples were collected from 136 COVID-19 patients (98 from Italy and 38 from Sweden) experiencing mild symptoms to critical disease ( Figure 1 , Table S1 and Table S2 ). Plasma from 108 historical negative controls collected before the SARS-CoV-2 pandemic were also analyzed. Plasma anti-RBD and anti-S antibody titers were measured by an in-house enzyme-linked immunosorbent assay (ELISA) 6 .

At the peak of the antibody response, 15-28 days after symptoms onset, anti-RBD IgM and IgA were increased in 77% (40/52) and 85% (44/52) of convalescent patients, respectively, but rapidly decreased between 1 and 3 months and were detected in less than 4.5% (2/44) and 11% (5/44) of patients tested between 6 and 15 months when assessing all COVID-19 subjects by cross-sectional analysis (Figures 2A, 2B, 2D Figures 2F, 2L) . The half-lives of anti-RBD and anti-S IgG antibody response estimated by a one-phase decay model were 134 and 113 days, respectively (Figures 2C, 2I) and were shorter in patients with mild/moderate (t 1/2 = 52 days and t 1/2 = 40 days) than severe/critical (t 1/2 = 372 days and t 1/2 = 239 days) disease (Figures S1A and S1B).

As a complementary approach, we analyzed the antibody titers from 42 patients who donated blood at two or more time points and estimated the half-lives of the RBD-and S-specific antibody response in IgM (t 1/2 = 71 days and 73 days), IgA (t 1/2 = 32 days and 28 days) and IgG (t 1/2 = 128 days and 90 days) (longitudinal analysis; Figure 3 ). No significant difference in the anti-S and anti-RBD IgG titers were observed between the paired samples (n = 11) containing a sampling time point between 181 and 300 days (6-10 months) and a second one between 301 and 452 days (10-15 months), confirming that specific IgG antibody titers reached a plateau phase after 6 months (p = 0.8984 and p= 0.3125, respectively; Figures 3C and 3F).

We further compared the antibody response induced from natural infection to that induced by one or two doses of the Comirnaty (Pfizer-BioNTech) vaccine (Table S3) The neutralizing activity against the G614 variant was measured by a microneutralization assay and was expressed as the neutralizing titers (≥ 1:10) which inhibit 90% of the virus infectivity (NT 90 ). Similar to the dynamic of the anti-RBD and anti-S antibodies, the median Figure 4C ). A one-phase decay model showed a rapid initial decay using both cross-sectional (t 1/2 = 70 days, Figure 4A ) or longitudinal analysis (t 1/2 = 44 days, Figure 4C ) which slow down to a plateau phase extending from around 4 up to 15 months. A decrease of NT 90 followed by a plateau phase was observed in both patients with mild/moderate and severe/critical diseases using cross-sectional analysis ( Figure S1C In order to evaluate if the convalescent patients could be protected from the circulating VOCs, we determined the cross-binding and cross-neutralizing activity against Alpha, Gamma, Beta and Delta variants using plasma collected at the early (15-106 days, median day of 24) and late (259-452 days, median day of 370) phase of convalescence from February to May 2021.

As described earlier, the IgG anti-RBD titers against wild-type RBD were higher in early ( Figure   4D ) compared to late convalescence ( Figure 4G ). Furthermore, a reduction in the level of IgG antibodies binding to Gamma and Beta RBD was observed using both plasma samples These data suggest that the plasma anti-SARS-CoV-2 antibody response and neutralizing activity decrease up to around 6 months but neutralizing activity is maintained in the majority of patients up to 15 months. Furthermore, plasma neutralizing activity was lower against Beta, Gamma and Delta variants, particularly 9-15 months after infection.

We analyzed 76 peripheral blood mononuclear cell (PBMC) samples collected from 54 patients Figure 5B ).

The S1 and S N M O peptide pool-specific T cells expressing interleukin-2 (IL-2) and/or interferon-gamma (IFN-γ) were measured by FluoroSpot assay. No or a negligible number of IL-2, IFN-γ, or IL-2/IFN-γ -producing T cells against the two peptide pools were detected in the negative controls. Overall, a T cell response against at least one of the SARS-CoV-2 peptide pools (S1, or S N M O protein derived) was detectable at a level above the cutoff in 95% (69/73) of the patient samples tested over the study period (Figures 6A-6C and S4A-S4C). When divided by groups based on the sample time points, specific T cells were detected in the majority of patient samples at 2-4 weeks (10/11, 91%) and 1-3 months (7/9, 78%), and more than 98% (52/53) of patient samples tested between 3 and 15 months.

Using cross-sectional analysis, the number of S1-specific IL-2, IFN-γ, and IL-2/IFN-γproducing T cells reached a peak between 3 and 6 months and was maintained up to 15 months except for IL-2/IFN-γ-producing T cells which significantly decreased at 366-452 days The numbers of S1 and S N M O peptide pool-specific IL2 and IL-2/IFN-γ -producing T cells were higher in severe/critical COVID-19 than mild/moderate patients between 6 and 15 months (p = 0.0044 and p = 0.0202 for S1 specific T cells, p = 0.0302 and p = 0.0427 for S N M O specific T cells) while no significant difference in S1 and S N M O specific IFN-γ -producing T cells was observed (p = 0.0610 and p = 0.1012, respectively) ( Figures S5 and S6 ).

The S1-specific T cell response measured in samples collected after one vaccine dose was equivalent to that observed in convalescent patients at 12-15 months when the specific T cell Finally, we have investigated the presence of all three arms of immunity for each individual who has been evaluated for SARS-CoV-2-specific adaptive immunity in all assays i.e., 1) IgM, IgG and IgA antibody response and/or neutralization activity, 2) RBD-specific IgG-producing B cells, and 3) S1 peptide or S N M O pool-specific IL-2 and/or IFN-γ-producing T cells ( Figure   S7 ). The majority of individuals (72%, 55/76) had three arms of immunity active against SAR-CoV-2, particularly from 2 to 15 months (80%, 48/60), highlighting the increase of memory B and T cells between 0 and 2 months and the long duration of the adaptive immune response.

Thus, SARS-CoV-2-specific memory B and T cells remained present in the majority (>95%) of patients followed up between 6 and 15 months but a reduction of the T cell response was observed at 12-15 months.

The magnitude, duration and quality of immunological memory are crucial for preventing reinfection. In this study, we extended our assessment of the longevity of the SARS-CoV-2- 7, 20 . In accordance with a previous study, the persistence of IgG antibody level was associated with disease severity and patients with milder disease appeared to have more rapid IgG anti-RBD antibody waning 21 . It has been reported that antibodies against SARS-CoV and Middle Eastern respiratory syndrome (MERS)-CoV, could still be detected 1-3 years after infection onset despite lack of re-exposure to this virus 22 . After a rapid decline of antibodies against SARS-CoV in the first two years, specific IgG have been detected in some patients up to 12 years after infection 23 suggesting that SARS-CoV-2-specific IgG antibodies may be present for a longer period in some individuals.

Functional neutralizing antibodies specific to SARS-CoV-2 (anti-S and anti-RBD) that are produced following infection or vaccination are considered important for viral neutralization and viral clearance 24, 25 . In the absence of definitive correlates of protective immunity, the presence of neutralizing antibodies against SARS-CoV-2 provides the best current indication for protection against reinfection 3,4,24 . As previously reported, the neutralizing ability of polyclonal plasma correlated positively with anti-S IgG or anti-RBD IgG 6, 19, 26 . Plasma neutralizing activity reached a plateau after 4-6 months and was maintained in the majority of patients up to 15 months which was consistent with a previous study showing no significant difference in anti-RBD IgG antibody level and plasma neutralizing activities against Wuhan strain between 6 and 12 months 7 . In addition, the two-phase pattern with an initial rapid decay of neutralizing antibodies which slowed down to a flat slope 27 might explain why early studies with shorter follow-up (2-4 months) reported a fast decline of the antibody response 28 . The long-lasting neutralizing activity might be caused by the accumulation of somatic mutations in IgG antibody genes and the production of antibodies with increased neutralizing potency 7, 27 . Although no significant difference in neutralization activity was observed beyond 6 months, it might be important to predict how the long-term neutralizing antibody decay will affect the clinical outcomes following both natural infection and vaccination. However, a recent study in fully vaccinated health care workers showed that the occurrence of breakthrough infection was more associated with the peak of antibody and neutralization titers induced by the vaccine rather than on the subsequent antibody decay suggesting that the degree of protection might depend more on the initial immune response and the generation of memory B and T cells 24 .

Although there is evidence that SARS-CoV-2 seropositivity is associated with protection against the same strain 29,30 , reinfection has been observed in some patients, which may represent non-durable protective immunity or infection with different variants [31] [32] [33] . Notable mutations identified in the S1 subunit of the Alpha (Del69-70, N501Y, P681H), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y), and Delta (L452R, T478K, P681R and occasionally E484Q) variants may lead to small changes in the structure of the S protein and RBD 8 leading to a higher infection rate and reduction of acquired immunity by neutralizing antibodies. As previously reported, we observed a substantial reduction of neutralization activity in the plasma of convalescent patients collected at early and more particularly, late phase of convalescence against the Beta, Gamma and to a lower extend Delta 34 . The N501Y mutation in the Alpha variant RBD does not alter the neutralizing ability of plasma antibodies from naturally infected individuals 35 while the Beta (B.1.351) and Gamma (P.1) carrying the N501Y and E484 mutations are more resistant to the neutralizing activity from convalescent and vaccine immune sera as well as neutralizing antibodies 7, 11, 36, 37 . The substitution at position E484 in the RBD of pseudovirus or recombinant virus is known to confer resistance to neutralization by convalescent human sera 12, 13 . The L452R mutation found in Delta was shown to impair neutralization by antibodies 38 Early T cell responses during COVID-19 have been correlated with rapid viral clearance and reduced disease severity 43 . In accordance with previous results, memory IL-2 and/or IFNγ−producing T cells specific to M, N nonstructural proteins, as well as S protein, were generated in the majority of convalescent patients following SARS-CoV-2 infection [43] [44] [45] [46] .

Functional SARS-CoV-2 specific T cells were detected at low level within 2 to 4 weeks from onset of symptoms and reached a peak between 3 and 6 months at levels similar to that of fully vaccinated individuals 4 . Specific IFN-γ producing Th1 CD4 + and cytotoxic CD8 + effector cells are considered important in supporting immunity against SARS-CoV-2 47 . Polyfunctional T cells, secreting more than one cytokine, are also typically associated with superior control of pathogens and may be important to prevent reinfection by SARS-CoV-2 48, 49 . The presence of dual positive IFN-γ and IL-2 S1-specific T cells that preferentially retain characteristics of both effector function and proliferative potential in vivo is indicative of strong and sustained Sspecific T-cell immunity. Similar to previous results 5 , a decrease in specific T cell response, and more particularly in dual positive IFN-γ and IL-2 S1-and S N M O-specific T cells, was observed after 6 months. Nevertheless, memory T cell were observed in the majority of the patients between 12-15 months and a longer follow-up period with more participants will be necessary to evaluate the sustainability of the T cell response beyond 15 months postinfection.

In conclusion, we observed that circulating memory B and T cells, and neutralizing antibodies 

The other authors declare no competing interests. See also Table S1-S3. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Harold Marcotte (harold.marcotte@ki.se).

RBD and S1-S2 proteins can be generated and shared on a collaborative basis.

All relevant data outputs are within the paper and its supplementation information.

Previously enrolled study participants were asked to return for a follow-up visit at the Disease severity was defined as mild (non-hospitalized), moderate (hospitalized, with lower respiratory tract infection, with dyspnea or not, but without oxygen support), severe (infectious disease/sub intensive ward with a need for oxygen and/or positive chest computed tomography scan, severe lower tract infections, with any oxygen support) and critical (intensive care unit (ICU) patients, intubated or with extracorporeal membrane oxygenation procedures) 6 .

The demographic and clinical characteristics of the patients for the entire 15 months follow-up are detailed in Table S1 and summarized in Table S2 Table S3 ).

The study in Italy was performed under the approval of the Institutional Review Board of Policlinico San Matteo (protocol number P_20200029440). The study in Sweden was approved by the ethics committee in Stockholm (Dnr 2020-02646). All participants provided written informed consent before participation in the study.

RBD-His protein was expressed in Expi293 cells and purified on Ni-NTA resin (#88221, Thermo Fisher) followed by size-exclusion chromatography on a Superdex 200 gel filtration column in PBS. 51 S1-S2-His (referred as S) protein from Wuhan-Hu-1 strain and RBD from variants (Alpha, Beta, Gamma, and Delta) were expressed baculovirus-free in High Five insect cells 52 and purified on HisTrap excel column (Cytiva) followed by preparative size exclusion chromatography on 16/600 Superdex 200 pg column (Cytiva) 53, 54 .

Levels of anti-S and anti-RBD IgM, IgA and IgG antibodies were determined by ELISA as previously described 6 AU/ml for anti-RBD IgM, > 0.08 AU/ml for anti-RBD IgA, and > 14.8 AU/ml for anti-RBD IgG, giving a specificity of 96% for IgM, 99% for IgA and 97% IgG.

For assessing the anti-RBD IgG binding activity against VOCs, plates were coated with RBD derived from Alpha, Beta, Gamma, and Delta (1.7 μg/ml) and 1:1600 dilution of selected serum or plasma collected from February to May 2021 at early (15-106 days, median day of 24) and late (259-452 days, median day of 370) phase of convalescence were added. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG and tetramethylbenzidine substrate were added as described above and the color reaction was stopped with 0.5M H 2 SO 4 after 10 min incubation. All samples were run in the same experiment and the results expressed as OD 450 .

As an internal control, a diluted in-house developed monoclonal antibody cross-binding all RBD variants was used.

SARS-CoV-2 strain G614 and VOCs (Alpha, Beta, Gamma, and Delta) were isolated from patients in Pavia, Italy and identified through next-generation sequencing method. A previously described microneutralization assay was used to determine the titers of neutralizing antibodies against SARS-CoV-2 strains in 171 samples 55, 56 . The neutralizing titer (NT 90 ) was the maximum dilution giving a reduction of 90% of the cytopathic effect. The cutoff for positivity was ≥1:10.

PBMCs were isolated from blood or buffy coat samples by standard density gradient centrifugation using Lymphoprep (Axis-Shield) and were cryopreserved and stored in liquid nitrogen until analysis. Total RNA was extracted from PBMCs by using RNeasy mini kit according to the manufacturer's protocol (Qiagen).

PBMCs were incubated for four days in RPMI-1640 medium with 10% FCS, supplemented with the TLR7 and TLR8 agonist imidazoquinoline resiquimod (R848, 1 µg/ml; Mabtech AB, Nacka, Sweden), and recombinant human IL-2 (10 ng/ml) for stimulation of memory B cells.

The number of B cells secreting SARS-CoV-2 RBD-specific IgG and total IgG were measured using the Human IgG SARS-CoV-2 RBD ELISpot PLUS kit (Mabtech AB) 6 . S1 and S N M O specific IFN-γ and IL-2-secreting T cells were detected using the Human IFNγ/IL-2 SARS-CoV-2 FluoroSpot PLUS kit (Mabtech AB) 6 . The plates pre-coated with capturing monoclonal anti-IFN-γ and anti-IL-2 were incubated overnight in RPMI-1640 medium containing 10% FCS supplemented with a mixture containing the SARS-CoV-2 peptide pool (scanning or defined pools), anti-CD28 (100 ng/ml) and 300 000 cells per well in humidified incubators (5% CO 2, 37°C). The SARS-CoV-2 S1 scanning pool contains 166 peptides from the human SARS-CoV-2 virus (#3629-1, Mabtech AB). The peptides are 15-mers overlapping with 11 amino acids, covering the S1 domain of the S protein (amino acid 13-685). The SARS- 

Mann-Whitney U test was used for comparisons between groups in anti-SARS-CoV-2 antibody levels, neutralization titers and numbers of specific memory B and T cells. Correlation analysis was performed using Spearman's rank correlation. The number of patients showing neutralization activity against variants was compared by Fisher's exact test. A Wilcoxon signed-rank test was used for comparison of paired samples. Continuous decay (linear regression), one-phase decay, two-phase decay, polynomial second order or log-normal of non-transformed and log2 transformed data were assessed with the best fitting statistical model chosen based on the F test. The half-lives of antibody and neutralization titers were estimated by a one-phase exponential decay model using non-transformed and log 2 transformed data, respectively. All analyses and data plotting were performed using GraphPad or R version 3.6.1. The results were expressed as the number of spots per 300,000 seeded cells after subtracting the background spots of the negative control. Symbols represent individual subjects. The cutoff value (dashed red line) was set at the highest number of specific B cell spots for the negative controls (> 12 spots / 300,000 cells). For each arm of immunity, the relative intensity of signals varies from no (grey), low (yellow) to high (dark green) signal. The gender (male: blue, female: red), age (from 22 (white) to 86 (dark pink) years), severity (from mild (light orange) to critical (dark orange)), number of signals (1, 2 or 3) , and the number of days after symptoms onset are shown. 

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