key: cord-0881140-5zerlx1r
authors: Paschold, Lisa; Klee, Bianca; Gottschick, Cornelia; Willscher, Edith; Diexer, Sophie; Schultheiß, Christoph; Simnica, Donjete; Sedding, Daniel; Girndt, Matthias; Gekle, Michael; Mikolajczyk, Rafael; Binder, Mascha
title: Rapid hypermutation B cell trajectory recruits previously primed B cells upon third SARS-CoV-2 mRNA vaccination
date: 2022-03-02
journal: bioRxiv
DOI: 10.1101/2022.03.01.482462
sha: 1f4f9b2269ca91e30042f2e63398cdc71381b3a0
doc_id: 881140
cord_uid: 5zerlx1r

High antibody affinity against the ancestral SARS-CoV-2 strain seems to be necessary (but not always sufficient) for the control of emerging immune-escape variants. Therefore, aiming at strong B cell somatic hypermutation - not only at high antibody titers - is a priority when utilizing vaccines that are not targeted at individual variants. Here, we developed a next-generation sequencing based SARS-CoV-2 B cell tracking protocol to rapidly determine the level of immunoglobulin somatic hypermutation at distinct points during the immunization period. The percentage of somatically hypermutated B cells in the SARS-CoV-2 specific repertoire was low after the primary vaccination series, evolved further over months and increased steeply after boosting. The third vaccination mobilized not only naïve, but also antigen-experienced B cell clones into further rapid somatic hypermutation trajectories indicating increased affinity. Together, the strongly mutated post-booster repertoires and antibodies deriving from this may explain why the booster, but not the primary vaccination series, offers some protection against immune-escape variants such as Omicron B.1.1.529. Brief summary Priming SARS-CoV-2 vaccinations generate antibodies from low-level matured B cells while the third vaccination strongly boosts somatic hypermutation potentially explaining different protection from immune-escape variants.

. Interestingly, even B cells with low or absent IGHV affinity maturation can generate antibodies that specifically recognize and neutralize the ancestral strain of SARS-CoV-2 (6, 11, (13) (14) (15) ). Yet, a continued evolution of the humoral response appears to take place over at least six months after infectioneven without re-infectionas demonstrated by sustained acquisition of IGHV somatic hypermutation despite waning antibody titers (16) (17) (18) (19) (20) (21) . There is emerging evidence that these rather mitigated B cell maturation dynamics may also be characteristic for vaccine-induced anti-SARS-CoV-2 immune responses (22, 23) .

With the advent of SARS-CoV-2 variants of concern, affinity to the ancestral strain's S protein (currently used in all licensed vaccines) does not necessarily predict antibody neutralization potency. Immune escape can affect clones with high affinity against the Page 4 of 33 ancestral strain but, depending on the targeted epitope, some clones also retain their neutralizing potency against variants of concern (24). This is in clear contrast to clones with low affinity to ancestral SARS-CoV-2 that constantly fail to neutralize variants (24) . This finding suggests that high affinity binding to the ancestral strain may provide some flexibility in compensating the effect of individual immune escape mutations (24) . Therefore, in times of emerging viral variants an optimal vaccination strategy should aim at inducing the highest possible level of affinity maturation through somatic hypermutation, even if the available vaccines are targeted at the ancestral strain.

In the study presented here, we used two cohorts of not previously infected patients to compare antibody levels and somatic hypermutation trajectories across a primary series of two standard vaccinations with those induced by a third "booster" vaccination using immune repertoire sequencing. We show that B lineage evolution is low after the priming vaccinations. In contrast, the maturation trajectories induced by the third vaccination is compatible with selective mobilization and germinal center recruitment of naïve but also previously matured memory B cell lineages to undergo fast and extensive somatic hypermutation. This considerable affinity maturation and the resulting high antibody titers may explain the increased protection of the third "booster" vaccination against variants such as the Omicron variant B.1.1.529.

For NIS635, we used a classical biobanking study (HACO) and a digital cohort study (DigiHero) with flexible recruitment of participants into different survey modules to obtain COVID-19 and vaccination data in a large cohort and to acquire relevant biological samples from subgroups of interest. In DigiHero, 514 participants donated blood and completed the survey of the COVID-19 module until December 2021. 4,670 participants completed the survey on the third SARS-CoV-2 "booster" vaccination until the same data cut. In HACO, data and sample collection had been completed in January 2021. Using this data, complete COVID-19 and vaccination histories could be deduced for all participants of NIS635. Details for all subcohorts are given in Table 1 .

In the DigiHero SARS-CoV-2 "booster" vaccination survey, the majority of participants indicated to have already received their third vaccination or to be planning to receive it shortly ( Figure 1A) . The time between completion of the primary vaccination series and the third booster vaccination is shown in Figure 1B . The majority of participants received BioNTech/Pfizer as their third vaccine ( Figure 1C ). The tolerability of the "booster" was roughly comparable to that of first and second SARS-CoV-2 vaccinations ( Figure 1D ). Most frequent side effects were local reactions, fatigue, and headache with comparable tolerability of both mRNA vaccines ( Figure 1E ).

Based on this survey, 15 participants without prior COVID-19 infection that planned a third vaccination within the next four weeks were randomly chosen and invited to donate blood before and after their third vaccination for antibody and B cell repertoire NGS analyzes.

Page 6 of 33 All biobanked samples indicated in Table 1 were tested for S1 and NCP antibodies by ELISA. Figure 2A shows the distribution of antibody levels for the different subgroups.

Participants vaccinated after infection (hybrid immunity) and participants after their third "booster" vaccination achieved the highest S1 antibody levels followed by previously uninfected participants that had only completed their primary vaccination series (Figure 2A ).

Only few individuals showed elevated NCP antibody levels despite having indicated no prior COVID-19 potentially pointing at unrecognized previous infection. All other participants showed antibody levels compatible with their infection/vaccination status. In all participants with matched pre-and post-vaccination samples, clear antibody increases were noted with highest levels after the third vaccination ( Figure 2B ).

Next, we compared antibody levels in participants with hybrid immunity to those after three vaccinations. Given the over-time decay in antibody titers both after infection and vaccination (16, 17, 20, (25) (26) (27) (28) , we included only participants in this analysis who donated blood in a standard interval of 2-4 weeks after the last vaccination dose. This analysis showed that antibody levels were similarly high in both subsets indicating that the third "booster" dose may mimic the hybrid-like response observed in individuals after infection and vaccination ( Figure 2C ).

Matched blood samples prior to and after the first/second or third vaccination were subjected to next-generation sequencing of the B cell receptor repertoire. The time points of blood collection are shown in Figure 3A . All patients included in this analysis had received mRNA vaccines; 8 of 15 participants received the BionTech/Pfizer vaccine as third vaccination.

There were no global differences in immune repertoire metrics such as diversity, richness or clonality across groups ( Figure 3B ). In addition, B cell repertoire somatic hypermutation rates of IGHV genes that reflect antigen-mediated affinity maturation were roughly identical Page 7 of 33 on the global immune repertoire level ( Figure 3C ). There were, however, trends in repertoire connectivity: Pre-vaccination B cell repertoires showed lowest connectivity between B cell clonotypes while samples taken after the third vaccination showed highest connectivity ( Figure 3D , lower part). B cell connectivity plots of two patients with representative connectivity levels are shown in the upper part of Figure 3D .

While global B cell repertoire metrics were rather stable across the studied subgroups, we hypothesized that the subrepertoire of B cells with known SARS-CoV-2 specificity may provide more insight into affinity maturation in response to vaccination. We, therefore, searched our set of immune repertoires for 3,195 known SARS-CoV-2 antibody sequences (29) to determine blood circulation of B cells carrying SARS-CoV-2 reactive B cell receptors.

1,147 thereof derived from neutralizing SARS-CoV-2 antibodies. Interestingly, bloodcirculation of such B cells appeared to be increased shortly after the first two vaccinations ( Figure 4A and 4B). After the third vaccination, we also noted increases as compared to the matched time point before the third vaccination ( Figure 4A and 4B ). Yet, in absolute numbers, the increase in blood circulation of these clones was lower than after the primary vaccination series.

In a next step, we determined somatic hypermutation rates of SARS-CoV-2 specific clones from matched pre-and post-vaccination samples. We reasoned that the rate of somatically hypermutated clones should increase with the number of applied vaccinations. Indeed, we found a continuous increase in the fraction of somatically hypermutated B cell clones within the SARS-CoV-2 specific repertoire from pre-vaccination samples to samples acquired after the third vaccination ( Figure 4C ). Interestingly, the rate of somatically hypermutated SARS-CoV-2 specific clones was lower after completion of the primary vaccination series than prior Page 8 of 33 to the third vaccination. This suggests that even "short-lived" mRNA vaccines trigger affinity maturation of B cells over months in line with recent data (22) . Although the somatic hypermutation load per SARS-CoV-2 specific sequence numerically increased in primed participants, it was dramatically boosted by the third vaccination ( Figure 4D ). This was especially observed for BCR sequences encoded by the IGHV3-21, IGHV3-23, IGHV3-53 and IGHV3-30-3 genes ( Figure 4E ) which have been already linked to S-reactive antibodies with exceptional neutralizing potency (12, (30) (31) (32) (33) (34) . The amount of hypermutation did not correlate with age ( Figure 4F ).

To be able to analyze individual maturation trajectories of B cells in the primary vaccination series versus upon third vaccination, we set out to identify developmental B lineages in all individual participants and to track them across the vaccination period. A B cell lineage is a group of clonotype-defined B cells that share a common V and J gene and a CDR3 sequence differing only in up to 10% of its amino acid positions (35) . While we did not detect expanding B cell lineages in a substantial proportion of participants receiving their primary vaccination series, we found expanding lineages in the majority of patients receiving their third vaccination ( Figure 5A ). The repertoire space taken up by these vaccine-induced expanding B cell lineages was substantially higher after the third vaccination than after completion of the primary vaccination series ( Figure 5B ). The stream plots in Figure 5C In the study presented here, we show complex B cell maturation trajectories induced by the third "booster" vaccination that by far exceeded the level of somatic hypermutation measurable directly after completion of the primary vaccination series. Interestingly, the strong mutational activity was essentially restricted to few lineages that were present and mutated already before the third vaccination and therefore likely involved in previously induced memory B cells that were once again recruited to the lymph node's germinal center Page 10 of 33 for further refinement. Notably, we observed high mutational rates especially in IGHV3-21, IGHV3-23, IGHV3-53 and IGHV3-30-3 genes that were linked to antibodies isolated from elite neutralizers that have previously shown neutralizing potency against variants of concern after infection with the ancestral strain (12, (30) (31) (32) (33) (34) . Interestingly, we did not observe any age-restriction for this maturation process indication that also older people benefit from a third vaccination although this needs further validation due to the limited size of the analyzed cohort. Since somatic hypermutation reflects affinity maturation, our data is not only well compatible with the strong increase in neutralization potential towards the ancestral strain induced by the third vaccination (45) , it also may explain why individuals after a recent third vaccination are usually protected from infection with the Delta variant that shows only few immune-evasive S protein mutations (46) (47) (48) . Since high affinity towards the ancestral strain is a prerequisite for variant neutralization, our data may also explain why the third Taken together, our data show that the primary vaccination series quickly generates antibodies from B cells that have only undergone low-level affinity maturation and may therefore not be protective for immune-escape viral variants such as Omicron B.1.1.529.

Our analyzes confirm the role of the third SARS-CoV-2 "booster" to generate affinitymatured clones and mobilize them for antibody production.

This study was registered as non-interventional study (NIS) at the Paul-Ehrlich-Institute Informed written consent was obtained and the study was approved by the institutional review board (approval number 2020-039). These samples were used for antibody and B cell repertoire next-generation sequencing analyzes. Table 1 summarizes all relevant participant numbers, their basic characteristics and biological samples used in NIS635. The study was conducted in accordance with the ethical principles stated by the Declaration of Helsinki. Informed written consent was obtained from all participants or legal representatives.

The collected plasma samples were isolated by centrifugation of whole blood for 15 min at 2,000xg, followed by centrifugation at 12,000xg for 10 min and stored at -80°C. Peripheral mononuclear cells (PBMC) were isolated by standard Ficoll gradient centrifugation.

Genomic DNA was extracted from PBMCs using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, USA).

Antibodies against the S1 domain of the spike (S) protein and the nucleocapsid protein (NCP) of SARS-CoV-2 were determined by Anti-SARS-CoV-2-ELISA IgA/IgG and Anti-SARS-CoV-2-NCP-ELISA kits from Euroimmun (Lübeck, Germany). Readouts were performed at 450 nm using a Tecan Spectrophotometer SpectraFluor Plus (Tecan Group Ltd., Männedorf, Switzerland).

Immunosequencing of B cell repertoires was performed as described in (54) . In brief, V(D)J rearranged IGH loci were amplified from 500 ng of genomic DNA using a multiplex PCR, pooled at 4 nM and quality-assessed on a 2100 Bioanalyzer (Agilent Technologies).

Sequencing was performed on an Illumina MiSeq (paired-end, 2 x 301-cycles, v3 chemistry).

Rearranged IGH loci were annotated using MiXCR v3.0.13 (55) and the IMGT 202011-3.sv6 IGH library as reference. Non-productive reads and sequences with less than 2 counts were discarded. All repertoires were normalized to 30,000 reads. Each unique complementaritydetermining region 3 (CDR3) nucleotide sequence was considered a clone. Broad repertoire metrics (clonality, diversity, richness) were analyzed as previously described (56) . IGHV genes were regarded as somatically hypermutated if they showed < 98% identity to the germline sequence.

We searched our IGH repertoires for validated SARS-CoV-2 antibody rearrangements with identical or highly similar CDR3 amino acid sequence (Levenshtein distance of ≤ 2) and identical IGHV-J gene usage as described in (13) . The validated SARS-CoV-2 antibody sequences were derived from CoV-AbDab accessed at 17 th December 2021 (29) and classified into 3,195 total SARS-CoV-2 binding sequences and 1,147 SARS-CoV-2 neutralizing sequences. A list of the target sequences is provided in Supplementary Table   1 .

To calculate network connectivity in BCR repertoires, we used the Levenshtein distance of all unique CDR3 amino acid sequences per repertoire using the imnet tool (https://github.com/rokroskar/imnet). Sequences with Levenshtein distance ≤ 3 were connected. For visualization as petri dish plots we used R package igraph and the fruchterman-reingold layout (57) . Data analysis and plotting was performed using R version (v4.1.2).

We identified overlapping B cell lineages in the pre-and post-vaccination time point per patient. A B cell lineage was defined as a group of B cell clonotypes that share a common V and J gene and a CDR3 sequence differing only in up to 10% of its amino acid positions (35) . Lineages and their evolution were visualised as stream plots with function plot.stacked from (https://www.r-bloggers.com/2013/12/data-mountains-and-streams-stacked-areaplots-in-r/). Data analysis and plotting was performed using R version (v4.1.2).

The herein reported sequence data set has been deposited at the European Nucleotide Archive (ENA) under the accession number PRJEB50803.

Differences between the four groups were analyzed by ordinary one-way ANOVA.

Differences between two groups were studied by unpaired, two-tailed student's t-test. All statistical analyses were performed using R version 4.1.2 and GraphPad Prism 8.3.1 (GraphPad Software, La Jolla, CA, USA).

This study was registered as non-interventional study (NIS) at the Paul-Ehrlich-Institute 

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We thank the participants of our DigiHero and HACO cohorts for their great support. 

Supplemental Table 1 : List of SARS-CoV-2 directed sequences used in our search algorithm.