After recovery from SARS-CoV-2 infection immunological memory could mediate a rapid immune response in case of reinfection


Until now, it was unclear whether a survived SARS-CoV-2 infection or COVID-19 leads to a persistent immunological memory and thus can protect against a new infection.

Several studies had shown that SARS-CoV-2 specific antibodies are only detectable for a few months in many people who have survived COVID-19 and may therefore only provide temporary protection against re-infection.

A research team at the Medical Center – University of Freiburg led by Dr. Maike Hofmann, Dr. Christoph Neumann-Haefelin and Prof. Dr. Robert Thimme has now been able to show: after recovery from SARS-CoV-2 infection, immune cells are formed which remain in the body and could mediate a rapid immune response in case of re-infection.

The Freiburg study was published in the online edition of the renowned scientific journal Nature Medicine on November 12, 2020.

“These so-called memory T-cells after SARS-CoV-2 infection look similar to those after a real flu.

We are therefore confident that the majority of people who have survived SARS-CoV-2 infection have some protection against re-infection with SARS-CoV-2,” explains Dr. Hofmann, a scientist at the Department of Medicine II at the Medical Center – University of Freiburg.

Professor Thimme, Medical Director of the Department of Medicine II, emphasizes how important a good translational research environment such as that at the Medical Center – University of Freiburg is in the current situation:

“In order to obtain robust research results within a few months, close networking between clinic and science at the highest level is a basic requirement: On the one hand, patients with COVID-19 are treated on our wards and continue to be cared for in a special outpatient clinic even after the infection has healed. On the other hand, our clinic has great expertise in the analysis of immune cells in viral infections such as hepatitis B and C.”

The Medical Center – University of Freiburg is not involved in the development of vaccines against SARS-CoV-2. However, Dr. Neumann-Haefelin, Head of the Gerok Liver Center at the University Hospital Freiburg, is optimistic:

“Our results suggest that immunity against SARS-CoV-2 can be achieved after an infection. Similarly, vaccines currently being tested in trials could provide significant protection against SARS-CoV-2″.

“The deciphering of complex immune responses has long been part of the research focus of the University and the Medical Center—University of Freiburg.

Thanks to the high scientific quality onsite, we can now make an important contribution to the corona pandemic,” says Prof. Dr. Norbert Südkamp, Dean of the Medical Faculty at the Albert-Ludwigs-University of Freiburg.

The rapid spread of the SARS-CoV-2 beta coronavirus has infected 19 million and killed over 700,000 people worldwide as of early August 2020. Infection causes the disease COVID-19, which ranges in presentation from asymptomatic to fatal. However, the vast majority of infected individuals experience mild symptoms that do not require hospitalization1.

It is critically important to understand if SARS-CoV-2–infected individuals who recover from mild disease develop immune memory that protects them from subsequent SARS-CoV-2 infections, thereby reducing transmission and promoting herd immunity.

Immunological memory is predominantly mediated by cells of the adaptive immune system. In response to most acute viral infections, B and T cells that can bind viral antigens through their antigen receptors become activated, expand, differentiate and begin secreting effector molecules to help control the infection.

Upon resolution of infection, approximately 90% of these virus-specific “effector cells” die, while 10% persist as long-lived “memory” cells2. Immune memory cells can produce a continuous supply of effector molecules, as seen with long-lived antibody-secreting plasma cells (LLPCs).

In most cases, however, quiescent memory lymphocytes are strategically positioned to rapidly reactivate in response to re-infection and execute effector programs imprinted upon them during the primary response. Upon re-infection, pathogen-specific memory B cells (MBCs) that express receptors associated with antigen experience and the transcription factor T-bet rapidly proliferate and differentiate into IgG+ antibody-secreting plasmablasts (PBs)3–5. Reactivated T-bet–expressing memory CD4+ T cells proliferate, “help” activate MBCs and secrete cytokines (including IFNγ) to activate innate cells2.

Meanwhile, memory CD8+ T cells can kill virus-infected cells directly through the delivery of cytolytic molecules6. These quantitatively and qualitatively enhanced virus-specific memory populations coordinate to quickly clear the virus, thereby preventing disease and reducing the chance of transmission.

To infect cells and propagate, SARS-CoV-2 relies on the interaction between the receptor binding domain (RBD) of its spike protein (S) and angiotensin converting enzyme 2 (ACE2) on host cells7. Multiple studies have shown that the majority of SARS-CoV-2 infected individuals produce S- and RBD-specific antibodies during the primary response, and RBD-specific monoclonal antibodies can neutralize the virus in vitro and in vivo8–10. Therefore, RBD-specific antibodies would likely contribute to protection against re-infection if expressed by LLPCs or MBCs.

To determine if the above hallmarks of immune protection from viral infection both form and persist in individuals that have experienced mild COVID-19, we assessed their SARS-CoV-2-specific immune responses at one and three months post-symptom onset. Herein we demonstrate that a multipotent SARS-CoV-2-specific immune memory response forms and is maintained in recovered individuals at least for the duration of our study. Furthermore, memory lymphocytes display hallmarks of protective antiviral immunity.

Return to immune homeostasis after mildly symptomatic COVID-19

To determine if immune memory cells form after mildly symptomatic COVID-19, we collected plasma and peripheral blood mononuclear cells (PBMCs) from 15 individuals recovered from COVID-19 (CoV2+) (UW IRB 00009810). The CoV2+ group had a median age of 47 and reported mild symptoms lasting a median of 13 days (E.D. Table 1).

The first blood sample (Visit 1) was drawn at least 20 days after a positive PCR test for SARS-CoV-2 and a median of 35.5 days post-symptom onset. We expect the primary response to be contracting and early memory populations to be generated at this time point, as viral load is cleared approximately 8 days post symptom onset11.

Participants returned for a second blood draw (Visit 2) a median of 86 days post-symptom onset so we could assess the quantity and quality of the long-lived memory populations (Fig. 1a). We compared these samples to samples collected at two time points representing a similar sampling interval in a group of 17 healthy controls (HCs).

All HCs were considered to have no prior SARS-CoV-2 infection based on having no detectable plasma SARS-CoV-2 RBD- or S-specific antibodies above three standard deviations (SDs) of the mean of historical negative (HN) plasma samples (E.D. Fig. 1).

We also included HN PBMC samples that were collected prior to the first human SARS-CoV-2 infection (2016-2019). We included these to control for the possibility that individuals in the HC group had been infected with SARS-CoV-2 (9/17 described having some symptoms associated with SARS-CoV-2 infection) despite their lack of detectable RBD-specific antibodies.

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Figure 1:
SARS-CoV-2-specific plasma antibodies at two memory time points.a) Study timeline. Range indicated by box and median indicated by line for each event. b) ELISA dilution curves and AUC for anti-RBD IgG (left), IgM (center), and IgA (right) from healthy control (HC) and previously SARS-CoV-2-infected (CoV2+) plasma samples at Visit 1 (V1) and Visit 2 (V2). Dashed line indicates mean + 3 SD of the HC AUC values. Each symbol is a different individual and is consistent throughout the figure. c) V2 CoV2+ AUC values were normalized to V1 samples run with V2 samples and AUC for each CoV2+ individual from V1 and V2 are paired. d) Percent inhibition of RBD binding to ACE2 by plasma at 1:10 dilution. e) Spearman correlation between percent RBD inhibition at a 1:10 plasma dilution and anti-RBD IgG AUC. f) CoV2+ percent RBD inhibition at 1:10 plasma dilution normalized and paired as in c). g) Spearman correlation between percent RBD inhibition at a 1:10 plasma dilution and percent virus neutralization by PRNT at a 1:80 plasma dilution. h) CoV2+ percent virus neutralization by PRNT at a 1:80 plasma dilution normalized and paired as in c). Statistical significance for unpaired data determined by two-tailed Mann-Whitney tests and, for paired data, by two-tailed Wilcoxon signed-rank tests. Error bars represent mean and SD (V1 HC n=15, V2 HC n=14 , V1 CoV2+ n=15, V2 CoV2+ n=14).

Populations of activated innate and adaptive immune cells expand in the blood during the primary response to SARS-CoV-2 infection12.

When an acute viral infection is cleared, the majority of these highly inflammatory cells either die or become quiescent memory cells such that the proportions and phenotypes of total immune cells are indistinguishable from those seen in pre-infection blood samples.

Consistent with resolution of the primary response, we found no differences in frequency of total monocytes, monocyte subsets or plasmacytoid dendritic cells among PBMCs between CoV2+ and HC individuals (E.D. Fig. 2).

We also found no differences in γδ or αβ CD3+ T cell frequencies (CD4+ or CD8+), nor in the cell cycle status, expression of molecules associated with activation, migration, function or proportions of various CD45RA− memory T cell subsets (E.D. Fig. 3). Together, these data demonstrate that the inflammatory response associated with acute infection had resolved by the Visit 1 time point and the early immune memory phase had commenced.

Mild COVID-19 induces persistent, neutralizing anti-SARS-CoV-2 IgG antibody

Humoral immune responses are characterized by a first wave of short-lived, low-affinity antibody-secreting PBs followed by a subsequent germinal center (GC) response that generates high-affinity MBCs and antibody-secreting LLPCs. LLPCs can maintain detectable serum antibody titers for months to many years, depending upon the specific viral infection13.

Thus, it is critical to distinguish the first wave of waning PB-derived antibodies from the later wave of persistent LLPC-derived antibodies that can neutralize subsequent infections, potentially for life.

We therefore first determined that CD19+CD20loCD38hi PBs were no longer present at elevated frequencies in CoV2+ individuals relative to HCs at Visit 1 (E.D. Fig. 4a).

Other measures of recent B cell activation in non-PB B cells include increased Ki67 expression (indicating cells have entered the cell cycle) and expression of T-bet14. There are small increases in both the frequencies of Ki67+ and T-bet+ B cells at the Visit 1 time point compared to HC, but not at the Visit 2 time point (E.D. Fig. 4b,​,c).c).

These data suggest that while PBs associated with controlling acute infection are no longer detectable in CoV2+ individuals at Visit 1, other B cell fates are still contracting. However, by Visit 2, these B cell phenotypes have returned to homeostasis (E.D. Fig. 4a–c).

Antibodies measured at Visit 1 might include contributions from short-lived plasmablasts, while those measured at Visit 2, long after PBs have contracted, represent contributions from LLPCs in the bone marrow.

We therefore examined the SARS-CoV-2-specific IgG, IgM and IgA antibodies at Visit 1 and Visit 215.

At Visit 1, 100% of CoV2+ individuals had plasma anti-RBD IgG levels 3 SDs above the mean of HCs, as measured by ELISA area under the curve (AUC), in accordance with studies showing 100% seroprevalence by day 1410 (Fig. 1b).

Additionally, 93% of CoV2+ individuals had anti-RBD IgM and 73% had anti-RBD IgA above this negative threshold. Almost all CoV2+ individuals possessed IgG (100%), IgM (100%), and IgA (93%) anti-spike antibodies above the threshold at Visit 1 as well (E.D. Fig. 4d).

Levels of anti-RBD and anti-spike binding were highly correlated for all isotypes (E.D. Fig. 4e). At Visit 2, all CoV2+ individuals maintained anti-RBD IgG levels above the negative threshold and 71% and 36% had maintained anti-RBD IgM and IgA, respectively (Fig. 1b).

Anti-RBD IgG levels decreased only slightly among CoV2+ individuals between time points and 36% of CoV2+ individuals had the same or increased levels at Visit 2. Anti-RBD IgM and IgA, however, decreased substantially from Visit 1 to Visit 2 (Fig. 1c, E.D. Fig. 4f).

As spike protein, and specifically the RBD, is key for viral entry into the cell, antibodies that target the RBD can be potent inhibitors of infection8,9. To determine whether CoV2+ individuals form and maintain neutralizing antibodies, we tested for SARS-CoV-2 neutralization indirectly using a cell-free competition assay (surrogate virus neutralization test, sVNT) and directly in a plaque reduction neutralization test (PRNT)16. CoV2+ plasma inhibited RBD binding to ACE2 significantly more than HC plasma by sVNT and RBD inhibition correlated strongly with anti-RBD IgG levels at both time points (Fig. 1d,​,e).e).

Further, RBD inhibition capacity was maintained or increased in the majority of CoV2+ individuals from Visit 1 to Visit 2 (Fig. 1f, E.D. Fig. 4g). Neutralization by PRNT correlated strongly with RBD inhibition at both time points (Fig. 1g, E.D. 4h) and was similarly maintained between visits (Fig. 1h).

By the latest time point in our study, 86% of CoV2+ individuals still had better RBD-inhibiting plasma than HCs and 71% had better neutralizing plasma (measured as above HC mean + 3 SDs). These data are consistent with the emergence of predominantly IgG+ RBD and spike-specific LLPCs that maintain detectable neutralizing anti-SARS-CoV-2 antibody to at least 3 months post-symptom onset.

Mild COVID-19 induces a sustained enrichment of RBD-specific memory B cells.

The presence of SARS-CoV-2-neutralizing antibodies three months post-symptom onset in CoV2+ individuals suggests GC-derived memory LLPCs have formed. GC-derived MBCs play a critical role in the formation of antibody secreting cells upon antigen re-exposure.

Therefore, we tested whether SARS-CoV-2-specific MBCs were also formed and maintained in CoV2+ individuals throughout the study time course.

We generated RBD tetramer reagents and used enrichment strategies to identify rare RBD-specific cells that are otherwise undetectable in bulk assessments17.

We confirmed specificity in RBD immunized mice and then used the RBD-tetramer to identify, enumerate and phenotype rare, RBD-specific B cells in our HN, HC and CoV2+ individuals (E.D. Fig. 5a,​,b;b; Fig. 2a). Gates used to phenotype RBD-specific B cells were defined on total B cell populations (E.D. Fig. 5c). At Visit 1, RBD-specific B cells were significantly expanded in CoV2+ individuals compared to HCs and their numbers were increased further at Visit 2 (Fig. 2a, ​,b).b).

The proportion and number of RBD-specific MBCs (defined by CD21 and CD27 expression) in CoV2+ samples was significantly greater than in HCs and increased from Visit 1 to Visit 2 (Fig. 2c,​,d,d, E.D. Fig. 5d). While RBD-specific B cells in HN samples had a similar proportion of MBCs as in CoV2+ samples, they contained substantially fewer cells.

In addition, RBD-specific MBCs were largely quiescent with very few expressing Ki67 (Fig. 2e, E.D. Fig. 5e). MBCs expression of class-switched B cell receptors (BCRs) is another marker of GC-derivation. We therefore assayed BCR isotype expression on RBD-specific MBCs and found enriched populations of IgA- and IgG-expressing MBCs in CoV2+ individuals at both time points (Fig. 2f–h, E.D. Fig. 5f).

Of note, while small numbers of RBD-specific MBCs were detected in controls, these cells were predominantly unswitched (IgM+ and IgD+), suggesting they may represent cross-reactive MBCs possibly generated in response to one of the human coronaviruses that cause 15% of common colds18–20.

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Figure 2:
RBD-specific MBCs form and persist in PBMCs post-mild COVID-19.
a) Representative gating of Live CD3−CD14−CD16− cells for SARS-CoV-2 RBD-specific cells (RBD tetramer+Decoy−) and b) number of RBD-specific B cells (RBD tetramer+Decoy−CD20+) from SARS-CoV-2-recovered (CoV2+) and healthy control (HC) PBMCs at Visit 1 (V1) and Visit 2 (V2). Gating strategy shown in Extended Data Figure 5c. c) Representative gating and d) Number of RBD-specific memory B cells (MBCs: CD21+CD27+/CD21−CD27+/CD21−CD27− populations outlined in red in c)(HN n=14, V1 HC n=12, V2 HC n=13 , V1 CoV2+ n=15, V2 CoV2+ n=14). e) Frequency of cycling (Ki67+) RBD-specific MBCs. f) Representative gating, g) frequency (HN n=14, V1 HC n=12, V2 HC n=13, V1 CoV2+ n=15, V2 CoV2+ n=14) and h) number of RBD-specific MBCs expressing the BCR isotypes IgD, IgM, IgA and IgG. i) Representative gating, j) frequency and k) number of RBD-specific MBCs expressing T-bet. Statistical significance determined by two-tailed, Mann-Whitney test (HC vs. CoV2+) and two-tailed Wilcoxon signed rank test (V1 vs V2). Error bars represent mean and SD (HN n=14, V1 HC n=15, V2 HC n=15, V1 CoV2+ n=15, V2 CoV2+ n=14 unless otherwise noted, 2 experiments).

An additional measure of antiviral MBC function is the graded expression of T-bet14. MBCs that express low-levels of T-bet are associated with rapid differentiation into secondary PBs that produce high affinity, viral-specific antibodies during a secondary infection21.

We found a higher proportion and number of T-bet+, and specifically T-betlo, RBD-specific MBCs in CoV2+ individuals compared with HCs at Visit 1 and the higher numbers were maintained at Visit 2 (Fig. 2i–k, E.D. Fig. 5g,​,h).h). T-bethi MBCs are considered to be recently activated and often found enriched during chronic infection21. Consistent with SARS-CoV-2 being an acute infection11, we found very few RBD-specific T-bethi MBCs in CoV2+ individuals at either memory time point (E.D. Fig. 5i).

Our data demonstrate that SARS-CoV-2 infection induces the generation of RBD-specific TbetloIgG+CD21+CD27+ “classical” MBCs likely derived from a GC22. Furthermore, numbers of these MBCs were not only maintained, but increased from one to three months post-symptom onset.

SARS-CoV-2 infection induces durable, functional spike-reactive CD4+ T cells

The presence of T-bet+ RBD-specific MBCs suggested that antigen-specific memory T cell responses were also likely to be elicited in CoV2+ individuals.

To enumerate SARS-CoV-2-specific memory T cells, total PBMCs from control or CoV2+ individuals were incubated with spike protein and expression of activation markers was assessed (Fig. 3a)23,24.

PBMCs from CoV2+ individuals at Visit 1 and 2 displayed robust re-activation of spike-specific CD4+ memory T responses, as measured by increased expression of ICOS and CD40L (two molecules associated with B cell help upon re-activation), while PBMCs from HC and HN individuals did not (Fig. 3a,​,b).b).

There were no significant differences in the numbers of responding cells in CoV2+ individuals between the two visits, suggesting spike-specific memory CD4+ T cells were maintained throughout the study (Fig. 3b).

Furthermore, greater numbers of CXCR5-expressing circulating T follicular helper (cTfh) cells25, which provide B cell help, were found within the population of S-specific ICOS+CD40L+CD4+ cells in CoV2+ individuals than in healthy controls at both visits (Fig. 3c). Together these data suggest that SARS-CoV-2-specific memory CD4+ T cells maintain the capacity to provide B cell help even at three months post-symptom onset.

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Figure 3:
Ex vivo reactivation of spike-specific CD4+ T Cells reveals durable and functional immune memory in SARS-CoV-2-recovered individuals.
a) Representative flow cytometry plots 20 hours after Vehicle control or Spike-stimulation of PBMCs from HC and CoV2+ individuals demonstrating T cell upregulation of CD40L and ICOS on CD45RA−CD4+ T cells. b) Enumeration of total CD40L+ICOS+ and c) CXCR5+CD40L+ICOS+ (cTfh) per 1e6 CD4+ T Cells and paired CoV2+ data from Visit 1 and Visit 2 represented as frequency of spike minus vehicle. d) Representative flow cytometry plots and e) number of CD69+ICOS+ CD4+ T Cells producing intracellular cytokines and number producing cytokine after incubation with spike minus number after incubation with vehicle. f) Relative distribution of effector cytokine production in memory T Cell compartments (CCR6+/− cTfh and non-cTfh) following ex vivo stimulation for 20 hrs; (IFN-y; blue) (IL-2; red) (IL-17A; yellow) from (d). g) Antigen-specific T cell proliferation of sorted CD4+ naive or memory T cells in control and CoV2+ PBMCs. Proliferation following 5-6 day co-culture with SARS-CoV-2 spike protein-pulsed autologous monocytes. h) Antigen-specific expansion represented as frequency of spike minus vehicle, CXCR3+CPDlow responding cells. i) Representative flow cytometry plots and j) quantification of spike-specific CD8+ T Cells in control and Cov2+ PBMCs stimulated with SARS-CoV-2 spike protein. a-h) Significance was determined by Kruskal-Wallis test correcting for multiple comparisons using FDR two-stage method. Adjusted p values are reported. i-j) Significance was determined by two-tailed, non-parametric Mann-Whitney tests. a-j) Data represented as mean and SD; Each symbol represents one donor. a-f, i-j) n=7 HN, n=14 HC, n=14 CoV2+(2 experiments). g-h) n=3 V1 HC, n=4 V2 HC, n=3 V1 CoV2+, n=4 V2 CoV2+ (2 experiments).

Memory CD4+ T cells produce cytokines within hours of activation, whereas naive T cells take days26.

We first examined cytokine production from activated CD4+ memory CXCR5− non-Tfh cells and CXCR5+ cTfh cells identified in the assay above (Fig. 3b).

S-specific CCR6+CXCR5+ cTfh cells, associated with IL-17 production, and a smaller population of CXCR3+CXCR5+ cTfh cells, associated with IFNγ production, were recently described in a predominantly mild to moderate cohort 30 days post symptom onset 27.

We therefore analyzed activated ICOS+CD69+ S-specific cells for expression of CCR6 and CXCR5 and then cytokine expression was examined in each population based on gating on a PMA positive control (Fig 3d, E.F. 6a).

Although multiple cytokines associated with Tfh function were assessed, only IFNγ, IL-17 and IL-2 cytokine producing cells were significantly expressed in activated S-specific memory CD4+ cells in CoV2+ individuals compared to HCs (Fig. 3d–f).

Small numbers of S-specific cells were measured in HCs after stimulation compared to vehicle alone that reflect previously described S-specific cross-reactivity20,28, but far greater responses were seen in the CoV2+ individuals (Fig. 3e).

Three months post symptom onset we found a higher frequency of CCR6− cTfh cells that produced Th1 cytokines, IFNγ and IL-2, suggesting a dominant Th1 response in CoV2+ individuals (Fig. 3f).

To further define the types of antigen-specific CD4+ memory T cells in CoV2+ individuals without relying on secretion of specific cytokines, we assessed memory CD4+ T cell proliferation in response to spike restimulation.

For this, we sorted CD45RA+ naive, CD45RA−CCR7+ central memory (Tcm) and CD45RA−CCR7− effector memory (Tem) T cells from HC or CoV2+ individuals (E.D. Fig. 6b), then measured the proliferative capacity of each sorted population following culture with autologous CD14+ monocytes and recombinant spike protein (Fig. 3g, ​,h;h; E.D. Fig 6c).

Only Tcm cells from CoV2+ individuals taken at both Visit 1 and Visit 2 displayed significant proliferation frequencies compared to HC samples, although substantial proliferative responses by Tem cells were observed in some CoV2+ individuals (Fig. 3h).

We also examined the expression of CXCR3 and CCR6 on S-specific, proliferated memory cells and found that the majority of cells that had proliferated, as measured by the dilution of cell proliferation dye (CPDlo) expressed CXCR3, in keeping with Type 1 cytokine production in the previous assay. Spike-specific Tcm, and potentially Tem, are therefore maintained throughout our study and have the ability to proliferate and re-populate the memory pool upon antigen re-encounter.

While much recent work has focused on antibodies and B cells, memory CD8+ T cells are uniquely positioned to kill virus infected cells through their directed expression of cytokines and cytolytic molecules. S-specific memory CD8+ T cells that persisted for three months after mild COVID-19 disease could be identified by expression of the activation marker CD69 and the cytokine IFNγ after overnight stimulation with spike (Fig 3i). Unlike CD4+ memory T cells, activated cytokine-expressing CD8+ T cells were significantly increased over vehicle controls in both control and CoV2+ groups (Fig. 3j).

Together, these data demonstrate that both CD4+ and CD8+ SARS-CoV-2-specific memory T cells are maintained and are able to produce effector cytokines after restimulation three months post-symptom onset in mildly symptomatic COVID-19 individuals.

Mild COVID-19-induced SARS-CoV-2-specific MBCs can express neutralizing antibodies

Since SARS-CoV-2 RBD-specific MBC and S-specific CD4+ cTfh were enriched in CoV2+ individuals after 3 months, we assessed whether these MBCs could produce neutralizing antibodies if they were reactivated by a secondary infection. To this end, we index sorted single RBD-specific B cells and sequenced the BCRs from 3 CoV2+ individuals at Visit 1 (E.D. Fig. 7a).

Of the class-switched (IgG+) RBD-specific classical MBCs (CD21+CD27+) we sorted, we randomly selected 7 to be cloned and expressed as IgG1 monoclonal antibodies (Fig 4a). This set of antibodies utilized a wide variety of heavy and light chains, had all undergone somatic hypermutation and were all unique clones (Fig. 4b, E.D. Table 2).

These antibodies were first expressed in small scale cultures. Transfection supernatants were assessed for antibody expression by IgG ELISA (E.D. Fig 7b) and specificity by RBD ELISA where all 7 showed strong binding to RBD (Fig. 4c). The first 4 antibodies cloned were expressed on a larger scale and purified.

The specificity of these purified antibodies for RBD was again confirmed by ELISA (E.D. Fig. 7c) and their ability to prevent SARS-CoV-2 infection was tested via PRNT assay. Two of the four tested (#202 and 203) showed strong virus neutralization (Fig. 4d), with IC50 values of 31 ng/ml for both (Fig. 4e). This was comparable to a previously published strongly neutralizing mouse antibody (B04) which was included as a positive control (IC50=7ng/ml)29.

Two of the RBD-specific antibodies were unable to inhibit virus infection, similar to a non-neutralizing mouse antibody (C02) and an irrelevant Plasmodium-specific human antibody. Three more monoclonal antibodies in addition to the 4 above were assessed for their capacity to inhibit RBD binding to the ACE2 receptor by sVNT assay (Fig. 4f).

Three of the seven were able to inhibit RBD binding to ACE2, similarly to a strongly neutralizing alpaca nanobody30. Interestingly, #203, which neutralized live virus, did not inhibit binding in this assay, while #202 both inhibited binding and neutralized the virus.

Overall 50% of the antibodies tested showed inhibitory activity by one or both of these methods. Thus, RBD-specific MBCs induced by SARS-CoV-2 infection are capable of producing neutralizing antibodies against the virus and could thus contribute to protection from a second exposure to SARS-CoV-2.

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Figure 4.
Generation of neutralizing antibodies by RBD-specific MBCs.
a) Flow plots of index sorted RBD-tetramer specific B cells (gating scheme in Extended Data Figure 7a). B cell receptors (BCRs) cloned from cells shown in red. b) Heavy and light chain gene usage, somatic hypermutation rate and VDJ junction sequence of cloned BCRs. c) Anti-RBD ELISA of culture supernatants from cells transfected to express one of the monoclonal antibodies compared to a known RBD-binding and neutralizing antibody (Ty1) and supernatant from untransfected cells (no transfection). d) Neutralization capacity of purified monoclonal antibodies as measured by PRNT. BO4 and CO2 are previously identified strong and weak neutralizing murine antibodies. e) IC50 values of antibodies calculated from PRNT. Dotted line represents the limit of detection. f) Inhibition of RBD-ACE2 binding by culture supernatants from antibody transfections (antibodies with high inhibitory capacity shown in red).

In the absence of a vaccine, natural infection-induced herd immunity could play a key role in reducing infections and deaths. For this to be possible, individuals that experience mild COVID-19 would need to develop and sustain protective immune memory.

Here, we found that individuals that recovered from mildly symptomatic COVID-19 had an expanded arsenal of SARS-CoV-2-specific immune mediators: neutralizing antibodies, IgG+T-betlo classical MBCs, circulating cytokine-producing CXCR5+ Tfh1 cells, proliferating CXCR3+ CD4+ memory cells and IFNγ producing CD8+ T cells that were maintained to at least three months post-symptom onset.

This study predicts that these recovered individuals will be protected from a second SARS-CoV-2 infection and, if so, suggests that Th1 memory should be the target of vaccine elicited memory.

Although long-lived immune memory can form to most viruses, some studies examining the longevity of the response to coronaviruses have suggested that this is not the case31–33. However, more recent studies, including our own, have examined memory time points when only LLPCs, and not short-lived PBs, are producing circulating antibodies.

Our study, along with three others clearly demonstrates elevated IgG+ RBD-specific plasma antibodies and neutralizing plasma are generated and maintained for at least 3 months post-SARS-CoV-2 infection34–36.

While antibodies reveal the contributions of LLPCs, functional virus-specific memory B and T cells can also be key to protective immune memory37. Although previous studies have measured the emergence of SARS-CoV-2-specific MBCs within a month of infection 27,38, we characterized SARS-CoV-2-specific MBCs at one and three months from symptom onset.

Our study revealed a prominent population of RBD-specific IgG+CD27+CD21+T-betlo MBCs, which has been associated in other infections with rapid differentiation into antibody-secreting PBs upon re-exposure5, effective antiviral responses39and long-lived protection3.

Furthermore, we found some of the RBD-specific MBCs at Visit 1 expressed BCRs capable of neutralizing the virus when expressed as antibodies. Since the numbers of these IgG+ RBD-specific MBCs were not only sustained, but continued to increase between one and three months, we predict they are GC-derived.

Thus, MBCs at three months would have undergone increased affinity maturation and we would expect an even higher percentage will be capable of producing neutralizing RBD-specific antibodies upon re-infection.

MBC reactivation requires interactions with memory CD4+ T cells, which reactivate MBCs through their expression of key molecules associated with T-B interactions including CXCR5, ICOS, CD40 and a variety of cytokines. SARS-CoV-2-specific CD4+ memory T cells in recovered individuals exhibited the capacity to express all of these molecules and to undergo robust proliferation upon re-exposure to spike protein. Notably, S-specific CD4+ memory T cells from CoV2+ individuals rapidly displayed increased levels of ICOS and CD40L on CXCR5+ and CXCR5− cells after stimulation as well as expression of Th1- and Th17- associated cytokines.

These results are consistent with another recent report of SARS-CoV-2- specific cTfh cells27, although they detected a high frequency of Th17-like cTfh cells, which could be due to the earlier time point they were examining as Th17 cells can develop into Th1 cells late in an immune response40.

The expression of IFNγ and IL-17 by these cells is notable as these cytokines are associated with class-switching to IgG and IgA isotypes, respectively 41,42. We also found cross-reactive memory B and T cells in healthy controls, as has been previously noted43.

It is difficult to measure their contribution to the expanded populations of SARS-CoV-2-specific cells we found in our CoV2+ cohorts, and therefore impossible to evaluate their protective capacity. However, we can conclude that mild COVID-19 induces an expanded population of functionally diverse memory lymphocytes compared to the cross-reactive pool present in our controls.

Studies of reinfection have yet to be done in humans, but macaques infected with SARS-CoV-2 were protected from rechallenge44. This further suggests that the immune memory induced by mild COVID-19 that we observed will be protective. While additional studies are needed to understand variability of responses in a larger cohort and to determine how long memory to SARS-CoV-2 infection is truly maintained, our work suggests that mild COVID-19 induces persistent immune memory poised for a coordinated, protective response to re-exposure that could contribute to herd immunity and curtailing this pandemic.

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More information: Isabel Schulien et al, Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells, Nature Medicine (2020). DOI: 10.1038/s41591-020-01143-2


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