Antibody feedback regulates immune memory after SARS-CoV-2 mRNA vaccination

Feedback inhibition of humoral immunity by antibodies was first documented in 19091. Subsequent studies showed that, depending on the context, antibodies can enhance or inhibit immune responses2,3. However, little is known about how pre-existing antibodies influence the development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity anti-SARS-CoV-2 monoclonal antibodies and subsequently two doses of an mRNA vaccine4–8. We found that the recipients of the monoclonal antibodies produced antigen-binding and neutralizing titres that were only fractionally lower compared than in control individuals. However, the memory B cells of the individuals who received the monoclonal antibodies differed from those of control individuals in that they predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations and showed altered receptor binding domain (RBD) target specificity, consistent with epitope masking. Moreover, only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The mechanism underlying these findings was examined in experiments in mice that showed that germinal centres formed in the presence of the same antibodies were dominated by low-affinity B cells. Our results indicate that pre-existing high-affinity antibodies bias germinal centre and memory B cell selection through two distinct mechanisms: (1) by lowering the activation threshold for B cells, thereby permitting abundant lower-affinity clones to participate in the immune response; and (2) through direct masking of their cognate epitopes. This may in part explain the shifting target profile of memory antibodies elicited by booster vaccinations9.

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Sample size
No a priori sample size calculations were performed. The sample size of 18 individuals (mAb recipients) derives from practical reasons in that it is purely based on how many study participants of the phase 1 study (NCT04700163) elected to subsequently receive mRNA vaccination, remained SARS-CoV-2 infection-naive throughout the study observation period, and could be recruited for serial blood donations at the Rockefeller University Hospital in New York City. Individuals from the vaccinated controls (n=31) were not de novo recruited and have previously been reported on extensively ( For mouse experiments (related to Fig. 4 and Ext. Data Fig. 7), the sample size of 6 individual animals per group was also not predetermined by statistical sample size calculations. Rather, it corresponds to a sample size that is generally accepted in the field, as it allows for rigorous hypothesis testing, simultaneously keeping the number of animals as small as possible while still being able to meet the scientific objectives (as per the 3R and ARRIVE guidelines).
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March 2021

Human research participants Policy information about studies involving human research participants Population characteristics
Participants in the monoclonal recipient group were healthy volunteers who had previously received a single dose of a combination of C144-LS and C135-LS, two human IgG1 neutralizing anti-RBD monoclonal antibodies (first characterized in Robbiani et al., 2020), in a phase 1, first-in-humans study to assess the safety and tolerability as well as the pharmacokinetics of the two antibodies (NCT04700163), and who subsequently got vaccinated with the initial two-dose regimen of either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccines against the wildtype (Wuhan-Hu-1) strain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of note, vaccinations were at the discretion of each individual participant and their health care providers and not part of our study design, which was purely observational in nature. Particpants were 43 (24-64) years old (median (range)), 5 out of 18 participants were female. 6

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The study was performed in compliance with all relevant ethical regulations and the protocols (CGA-1015 and DRO-1006) for studies with human participants were approved by the Institutional Review Board of the Rockefeller University.
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Clinical trial registration NCT04700163

Data collection
The study "A Phase 1, Open Label, Dose-escalation Study of the Safety and Pharmacokinetics of a Combination of Two Anti-SARS-CoV-2 mAbs (C144-LS and C135-LS) in Healthy Volunteers" (NCT04700163) was conducted at The Rockefeller University between January 11, 2021 and February 2, 2022.
Outcomes NCT04700163 was conducted to assess the safety and tolerability, as well as the pharmacokinetics of C144-LS and C135-LS, with adverse events and phamacokinetic properties of the infused antibodies as its primary and secondary outcomes.
However, the study presented here explicitly does not report on the pre-defined endpoints of NCT04700163. Instead, the data presented in this manuscript merely represents an observational study of the immune response to vaccination in participants of NCT04700163, which does not constitute a pre-specified outcome of NCT04700163.

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March 2021
For mouse experiments, popliteal lymph nodes from mice 11 days after immunization were isolated and collected in FACS buffer (1x PBS, 2% FBS, 2 mM EDTA). Single cell suspensions of the pooled popliteal lymph node samples from each respective mouse were subsequently processed as described. For the mouse experiments, GC B cell abundance was not a limiting factor for cell sorting. Sorting efficiencies (based on the same calculation as above) were slightly lower (between 30 to 80%), with the notable difference that only IgK-specific light chain primers were used.
For mouse experiments, gating was as detailed in Ext. Data Fig. 7a. Briefly, single live cells were gated to only include cells negative for staining with anti-CD4-APC-eFluor780, anti-CD8a-APC-eFluor780, anti-NK1.1-APC-eFluor780, anti-F4/80-APC-eFluor780 and and Zombie NIR to exclude dead and irrelevant cell populations. Next, cells positive for staining with anti-CD45R/B220-BV605 were considered B cells. B cells with MFIs <1000 for staining with anti-CD38-PB/BV421 and positive for staining with anti-GL7-FITC and anti-CD95-PE-Cy7 were considered GC B cells. Among those, cells with MFIs higher than 500 for RBD-A647 and 1000 for RBD-PE were deemed RBD-binding. Cell sorting was done on cells in the GC B cell gate agnostic of binding to RBD. RBD-binding status of single-sorted cells was established post-factum through index sorting data, using the same gating as in Ext. Data Fig 7a and b.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.