Researchers fuond a subset of individuals that heal quickly while sustaining virus-specific antibody levels after COVID-19


One of the pressing questions about COVID-19 remains: How long does immunity last?

One key indicator of immunity is the presence of virus-specific antibodies.

Previous studies have provided conflicting accounts about whether people who have recovered from infection can sustain potentially-protective antibodies or not.

A new study led by investigators from Brigham and Women’s Hospital examined blood samples and cells from patients who had recovered from mild to moderate COVID-19 and found that while antibodies against the virus declined in most individuals after disease resolution, a subset of patients sustained anti-virus antibody production several months following infection.

These antibody ‘sustainers’ had a shorter course of symptoms, suggesting that some individuals who recover from COVID-19 faster may be mounting a more effective and durable immune response to the virus. Results are published in Cell.

“We’ve found a subset of individuals that heal quickly while sustaining virus-specific antibody levels after COVID-19,” said Duane Wesemann, MD, Ph.D., an immunologist and associate physician in the Brigham Division of Allergy and Clinical Immunology and an associate professor at Harvard Medical School.

“The kind of immune response we’re seeing in these individuals is a bit like investing in an insurance policy – it’s the immune system’s way of adding a potential layer of protection against future encounters with the virus.”

The Wesemann lab studies the entire set of antibodies a host’s immune system produces and how they learn to recognize pathogens. In the spring of 2020, the team turned its attention to the COVID-19 pandemic and the immune response of people who become infected.

They are eager to understand the nature of the antibody response to the virus. To this end, the team recruited and enrolled 92 people in the Boston area who had recovered from COVID-19 between March and June of 2020.

Five of the individuals were hospitalized but all others recovered at home. The team collected and analyzed blood samples monthly, measuring a range of antibodies, including immunoglobulin-G (IgG), against the virus that causes COVID-19.

They split the cohort into two groups – those that sustain virus-specific IgG levels over several weeks, and those that lose them. The team analyzed these groups and potential connections they had to clinical and other immunological data.

The team found that IgG levels against the virus tended to decline substantially in most individuals over the course of three to four months. However, in about 20 percent of individuals, antibody production remained stable or enhanced over the same time period.

The team found that these “sustainers” had symptoms for a significantly shorter period of time compared to “decayers” (average of 10 days versus 16 days). Sustainers also had differences in memory T cell populations and B cells, two types of immune cells that can play a key role in immune memory and protection.

“The data point to a type of immune response that’s not only adept at handling viral disease by leading to a swift resolution of symptoms, but also better at producing cells that can commit to longer term production of anti-virus IgG antibodies,” said Wesemann.

“Figuring out how these individuals are able to support longer-term antibody production is relevant to COVID-19, and will also have important implications for our understanding of the immune system in general.”

Since its discovery in Wuhan in 2019, the causative agent of COVID-19, the SARS-CoV-2 virus (Zhu et al., 2020), has become a major global public health problem. A better understanding of immune responses induced by SARS-CoV-2 is urgently needed to help control the infection.

Several studies have shown that the neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery (Beaudoin-Bussières et al., 2020; Long et al., 2020; Prévost et al., 2020; Robbiani et al., 2020; Seow et al., 2020).

Although a good correlation between the presence of Spike (S)-specific antibodies and the capacity of plasma from infected individuals to neutralize viral particles was reported, recent data looking at individual immunoglobulin (Ig) isotypes revealed a stronger correlation between the decrease in S-specific IgM antibodies and loss of neutralization compared to S-specific IgG and IgA antibodies, suggesting that IgM play an important role in the neutralization activity of plasma from individuals who suffered from COVID-19 (Beaudoin-Bussières et al., 2020; Prévost et al., 2020).

To better understand the relative contribution of S-specific IgM, IgA and IgG antibodies in SARS-CoV-2 neutralization, we selectively depleted each Ig isotype from plasma obtained from 25 convalescent donors and assessed the impact of depletion on the capacity of the plasma to neutralize SARS-CoV-2 pseudoviral particles and wild type infectious SARS-CoV-2 viral particles.

Ig depletion
Demographic information of the 25 convalescent donors (21 males, 4 females, median = 45 days after symptoms onset), who were diagnosed with or tested positive for SARS-CoV-2 with complete resolution of symptoms for at least 14 days before sampling are presented in Table 1.

Selective depletion of IgM, IgA or IgG was achieved by adsorption on isotype-specific ligands immobilized on Sepharose or agarose beads, starting with a five-fold dilution of plasma (see details in Stars Methods). The depletion protocols permitted to efficiently deplete each isotype while leaving the other isotypes nearly untouched, as measured by ELISA (Fig 1A-C).

Depletion of IgG had a much higher impact on the total level of SARS-CoV-2 RBD antibodies than IgM and IgA depletion (Fig 1D), although RBD-specific antibodies of each isotype were selectively removed by the depletion (Fig. 1E-G).

The impact of IgG depletion on the level of total antibodies against the full S glycoprotein expressed on 293T cells (measured by flow cytometry) was also noticeable (Fig. 1H) whereas isotype-specific detection of full S antibodies by flow cytometry confirmed the efficacy of selective depletion (Fig. 1I-K).

IgM, IgA and IgG depletion in plasma samples from convalescent donors.
(A-C) Efficacy of the specific isotype depletion assessed by ELISA for total IgM, IgA and IgG. All plasma samples were diluted 5-fold prior to depletion; (A) IgM concentration in non-depleted, IgM-depleted, IgA-depleted and IgG-depleted plasmas, measured using an anti-human IgM (μ-chain specific) as capture antibody; (B) IgA concentration measured on the same plasmas using anti-human IgA (α-chain specific); (C) IgG concentration measured using anti-human IgG (γ-chain specific). (D-G) Efficacy of SARS-CoV-2 specific antibody depletion assessed by SARS-CoV-2 RBD ELISA; (D) Level of total (pan-Ig) anti-SARS-CoV-2 RBD-specific antibodies in non-depleted, IgM-depleted, IgA depleted and IgG-depleted plasmas; (E) Level of IgM-specific anti-RBD; (F) Level of IgA-specific anti-RBD; (G) Level of IgG-specific anti-RBD. (H-K) Efficacy of full S glycoprotein-specific antibody depletion measured by flow cytometry; (H) Level of total (pan-Ig) anti-SARS-CoV-2 S-specific antibodies in non-depleted, IgM-depleted, IgA-depleted and IgG-depleted plasmas; (I) Level of IgM-specific anti-S; (J) Level of IgA-specific anti-S; (K) Level of IgG-specific anti-S. Asterisks indicate the level of statistical significance obtained by a Dunn’s test; **** p<0.0001.

Neutralizing activity of depleted plasma

We then evaluated the capacity of non-depleted and isotype-depleted plasma samples to neutralize pseudoviral particles expressing the S glycoprotein from SARS-CoV-2 (Prévost et al., 2020) (Star Methods). Depletion of IgM, IgA or IgG all resulted in a significant decrease of neutralization compared to non-depleted plasma (Fig. 2A-D).

However, the loss of neutralization activity was much more pronounced in IgM- and IgG-depleted plasma with a 5.5 and 4.5 fold decrease in mean ID50 compared to non-depleted plasma respectively, than in IgA-depleted plasma where a 2.4 fold decrease only was observed (Fig. 2E).

To evaluate whether the impact of isotype depletion on neutralization could be extended beyond pseudoviral particles, we tested plasma from eight donors in microneutralization experiments using fully infectious SARS-CoV-2 viral particles, as described in the Star Methods.

The neutralizing potency of plasma was greatly reduced following IgM and IgG (4.0 and 2.9 fold respectively) but not IgA (no decrease) depletion (Fig. 2F and G). Despite the limited number of samples tested with the live virus, the impact of IgM and IgG depletion on neutralization was similar to that observed with the same samples in the pseudoviral particles neutralization assay (Fig. 3A-C).

This data not only confirms the role of IgG in neutralizing activity of convalescent plasma but also highlights the important contribution of IgM with respect to neutralization activity.

Role of IgM, IgA and IgG in neutralization.(A) Comparison of the SARS-CoV-2 pseudoviral inhibitory dilution (ID50) of all plasma samples. (B-D) ID50 of plasma from each convalescent donor before and after (B) IgM, (C) IgA and (D) IgG depletion. (E) Fold decrease (isotype-depleted versus non-depleted plasma) in ID50 measured by SARS-CoV-2 pseudovirions neutralization. (F-G) Microneutralization assay using infectious wild type SARS-CoV-2 performed on non-depleted and isotype-depleted plasma from 3 donors; (F) Mean percentage of infection observed with plasma from the 3 donors and (G) Fold decrease (isotype-depleted versus non-depleted plasma) in ID50 measured by microneutralization of wild type SARS-CoV-2 virions. Asterisks indicate the level of statistical significance obtained by a Wilcoxon signed rank test, n.s. not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Neutralizing capacity of eight convalescent plasma using pseudoviral particles or microneutralization with infectious wild type SARS-CoV-2 virus.
ID50 obtained by (A) virus microneutralization assay or (B) pseudoviral particles neutralization assay for non-depleted or isotype-depleted plasma of eight convalescent donors. (C) Spearman correlation and linear regression fitting between the ID50 obtained by microneutralization and pseudoviral particles neutralization assays. Dashed lines indicate the 95% confidence interval of the linear regression fitting. Non-depleted plasmas are shown in black, IgM-depleted in blue, IgA-depleted in red and IgG-depleted in green. Asterisks indicate the level of statistical significance obtained by a Wilcoxon signed rank test, n.s. not significant; **p<0.01; ****p<0.0001.


Our findings detailing the important role of IgM in the neutralizing activity of convalescent plasma has several implications. First, although the therapeutic efficacy of convalescent plasma for the treatment of COVID-19 patients remains to be established, it is likely that neutralizing antibodies will play a role.

Because SARS-CoV-2 specific IgM antibodies rapidly decrease after disease onset (Beaudoin-Bussières et al., 2020; Prévost et al., 2020; Robbiani et al., 2020; Seow et al., 2020), the collection of convalescent plasma with maximal neutralizing activity should be performed early after disease recovery.

Second, our results suggest that caution should be taken when using therapeutics that impair the production of IgM. Anti-CD20 antibodies (B cell-depleting agents) are used to treat several inflammatory disorders. Their use is associated with IgM deficiency in a substantial number of patients, while their impact on IgG and IgA levels is more limited (Kridin and Ahmed, 2020).

In line with our data, recent studies reported that anti-CD20 therapy could be associated with a higher susceptibility to contract SARS-CoV-2 and develop severe COVID-19 (Guilpain et al., 2020; Hughes et al., 2020; Safavi et al., 2020; Schulze-Koops et al., 2020; Sharmeen et al., 2020; Sormani et al., 2020).

Whether this is associated to the preferential depletion of IgM-producing B cells by these treatments (Looney et al., 2008) remains to be shown. Nevertheless, our results suggest that IgM levels should be investigated as a biomarker to stratify patients on immunosuppressive therapies at higher risk for COVID-19.

In summary, our results extend previous observations showing a strong correlation between neutralization potency and the presence of RBD-specific IgM (Beaudoin-Bussières et al., 2020; Perera et al., 2020; Prévost et al., 2020; Seow et al., 2020). It is intriguing that IgM represents about only 5% of the total antibodies in plasma (Wang et al., 2020), yet plays such an important role in SARS-CoV-2 neutralization.

Whether this is due to the enhanced avidity provided by its pentameric nature remains to be formally demonstrated but is in agreement with recent work demonstrating that dimeric antibodies are more potent than their monomeric counterpart (Wang et al., 2020).

The possible establishment of long lived IgM-producing B cells that might contribute to long term immunity of recovered patients has been suggested (Brouwer et al., 2020; Newell et al., 2020). However, how plasma neutralization evolves over prolonged periods of time and the specific role of IgM in this activity remains to be determined.

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More information: Yuezhou Chen et al, Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production, Cell (2020). DOI: 10.1016/j.cell.2020.10.051


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