Detecting more and less abundant antibody clones in serum by LC-MS-based Fab profiling is challenging as all molecules are alike, leading to extreme spectral congestion following electrospray ionization. To reduce spectral congestion, we implemented gas-phase fractionation using proton transfer charge reduction in a data-independent analysis manner (DIA-PTCR). Employing just five isolation windows over a limited m/z region was found to be sufficient to cover the total Fab distribution. This gas-phase fractionation, when also combined with reduced noise filtering, increased the number of clones detected within a single run from 1043 to 2063 (+100%). Importantly, quantitative data on antibody abundances could be retained as required for repertoire profiling. We conclude that DIA-PTCR is beneficial for LC-MS-based antibody repertoire profiling.

Immunoglobulin A (IgA) is the most abundant antibody in humans, with high concentrations in both mucosae/secretions and circulation. While mucosal IgA has been studied extensively, characterization of human IgA in serum and its distinctive functions lags behind. Circulatory IgA is regularly assumed to be monomeric, despite some reports describing a minor population of J-chain-coupled dimers. Here, we first charted the compositional landscape of human serum IgA in individual healthy donors. In addition to the expected predominating monomers, we consistently observed J-coupled dimers, even representing ~30% of one donor's total serum IgA. To determine whether these structurally distinct populations were also clonally distinct, we employed mass spectrometry-based IgA1 clonal profiling in sera of two donors. Our data revealed the majority of IgA1 clones are present solely as monomers, with a smaller number exclusively dimeric. Strikingly, a third population of IgA1 clones is present in circulation as both monomers and J-coupled dimers. In fact, these shared, structurally promiscuous IgA1 clones dominated both individuals' serum IgA1 clonal repertoires. Our findings suggest every unique IgA1 clone could potentially be produced as a variable mixture of monomers and J-coupled dimers, but what exactly determines this ratio requires further investigation. This finding is important, as monomers and J-coupled dimers have distinct characteristics in antigen binding, receptor activation, and clearance from circulation, with several reports highlighting, for instance, the enhanced neutralization capacity of dimeric IgA. As we show here, the human immune system is not merely capable of producing both forms but apparently prefers doing so in parallel.

Objectives:The early development of humoral immunity is important for long-term protection of the newborn. Here, we set out to discern these infant-produced antibodies from the vast background of maternal antibodies, which is challenging but essential to shed light on the infant-produced repertoire. Methods:Using IgA1 and IgG1 antibody clonal repertoire analysis by mass spectrometry, we compared matched maternal serum, maternal milk, and infant serum samples, both at birth (T1) and at 7-11 weeks after delivery (T2) in four mother-infant dyads. Results:We observed for both IgA1 and IgG1 unique infant-produced antibody repertoires at T2. For IgA1 at T2, no substantial clonal overlap was found between infant serum and breastmilk. The serum IgG1 clonal repertoires were highly alike at birth for mother and infant, but at T2, the contribution of the maternal clonal population in the infant had been drastically reduced, and a large portion of the T2 IgG1 repertoire originated from the infant. Conclusions:Newborns produce their own antibody repertoires as early as a few months after birth. From this small study, no convincing evidence is found for transfer of milk antibodies into the infant circulation.

Out of all human antibodies, IgG4 stands out by its ability to dissociate in half-molecules and reassemble forming novel bivalent antibodies. Although this so-called Fab-arm exchange mechanism is acknowledged, detailed analysis of whether all IgG4 clones in serum are involved in such processes is not known. Here, by introducing a liquid chromatography-mass spectrometry-based approach enabling the analysis of serum IgG4 clonal repertoires, we show that widespread Fab-arm exchange occurs in serum of healthy donors and leads to a massive explosion in the molecular diversity of the IgG4 clonal repertoire. These findings provide new insight into IgG4, which plays a critical role in allergy, autoimmunity and vaccination settings, and may also impact the use of IgG4 as scaffold for therapeutics. Among the antibody subclasses, IgG4 is unique in its capability to fragment-antigen-binding (Fab)-arm exchange, a process that renders IgG4 bispecific for antigen binding. Here authors analyse serum IgG4 clonal repertoires of healthy human donors to show that Fab-arm exchange is widespread and affects all IgG4 clones.

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and cartilage/bone destruction. RA affects the synovial joints, the synovial lining, and the permeability of the synovium. As the latter is of central relevance for the distribution of systemically delivered therapeutics into synovial fluid (SF), we here assessed the protein composition of paired plasma and SF of patients diagnosed with RA at three distinct levels of depth using mass spectrometric approaches: the "total" proteome, the "total" immunoglobulin G1 (IgG1) antibody repertoire, and the RA-specific anticitrullinated protein IgG1 autoantibody repertoire. The SF proteome was found to be dominated in numbers and concentration by plasma proteins, although we additionally detected several cartilage- and neutrophil-derived proteins of lower abundance. Strikingly, the plasma proteins were not only qualitatively reflected in SF but also quantitatively, independent of their size and/or other biochemical features. Also, the synovial "total" IgG1 and autoreactive anticitrullinated protein antibody IgG1 repertoire highly resembled the IgG1 repertoires detected in plasma within the same patient. Our comprehensive multilayer data thus reveals that the proteome, including the dominant, most abundant (auto)antibody clones, present in SF of RA patients is a direct reflection of the proteome present in blood, spiked by the local (immune) processes within the RA joint. We thus conclude that proteins directly pass from blood into SF of these joints without substantial bias. These findings thereby not only exemplify the use of in-depth multilayer proteome analyses to revisit basic concepts underlying RA pathology and to monitor the local (immune) processes destructive to cartilage but also provide evidence indicating that (protein-based) therapeutics may equally enter SF of swollen joints and that pharmacokinetic analyses of such therapeutics in blood are directly relevant to the synovial compartment.

BACKGROUND:Haemolytic disease of the fetus and newborn (HDFN) is caused by maternal alloantibodies, often targeting the D antigen on fetal red blood cells. Maternal immunization is preventable with timely administration of anti-D polyclonal antibodies (pAbs). Although the mechanisms of action for pAbs are not completely known, clinical efficacy has been suggested to be associated with afucosylated anti-D IgG and strong Fc-receptor-mediated antibody-dependent cellular cytotoxicity (ADCC). Anti-D pAbs are derived from hyperimmunized individuals, which makes the supply expensive and donor dependent. Monoclonal antibodies (mAbs) offer an alternative, but none have successfully prevented HDFN; some of them even enhanced alloimmunization. Despite lacking food and drug administration / European medicines agency (FDA/EMA) approval, two mAbs-Rhoclone™ and Trinbelimab (TBL) (Anti D®)-are widely used in low/middle-income countries. STUDY DESIGN AND METHODS:Here we investigated functional and structural properties of these mAbs, including epitope mapping, glycan composition, and de novo sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). Then, antibody engineering was employed to enhance ADCC potential. RESULTS:Both Rhoclone (Rho) and TBL recognized RhD-epitope 5.5. Amino acid sequencing revealed these mAbs to be identical at the protein level, and that TBL had lower fucosylation (86%) than Rho (96%). Both mAbs had lower ADCC activity than anti-D pAb Rhophylac®. ADCC performance was correlated with fucosylation levels: afucosylated engineered anti-D > pAbs > TBL > Rho ≈ fucosylated anti-D control. Glycoengineered versions with low fucose showed markedly enhanced natural killer cell (NK)-cell-mediated ADCC. DISCUSSION:Afucosylation of anti-D monoclonals mimics polyclonal anti-D and enhances their ADCC. Future efforts should focus on determining if these functional differences translate to clinical efficacy.

Antibodies are key molecular elements of the human immune system and account for an increasingly large proportion of therapeutics. Next to the well-studied and explored IgG1, several other classes (e.g. IgA, IgM) and sub-classes (e.g., IgG2, IgG3 and IgG4) exist in humans. In particular IgG4 is worth a closer examination as it has unique natural properties and is regularly used as a scaffold in biologicals. IgG4 stands out from the other IgG by its ability to dissociate and form two half-molecules which can interchange with those of other clones to form novel bivalent antibodies. Detailed analysis of endogenous IgG4 so far has been hampered by a lack of analytical methods to dissect and analyze IgG4 molecules with clonal resolution. Here, we present an LC-MS-based approach enabling the analysis of IgG4 repertoires with clonal resolution, which we used to monitor endogenous serum IgG4 repertoires from seven healthy donors. Most strikingly, our data reveal the combinatorial explosion in diversity of the serum IgG4 clonal repertoire. This phenomenon is explained by the stochastic behavior of Fab-arm exchange, making virtually each IgG4 molecule in serum bispecific. Although the endogenous IgG4 clonal repertoire is therefore extremely diverse, we demonstrate that this IgG4 repertoire persists over time within an individual healthy donor for more than one year. This newly established method now enables repertoire analysis for IgG4, which plays a critical role in a plethora of disease settings including allergy, autoimmunity and vaccination. ### Competing Interest Statement The authors have declared no competing interest.

Antibodies play a pivotal role in the immune defense and long-term immunity. Yet, while several studies have highlighted the persistence of antigen-specific antibody responses, it is unclear whether this stems from the continuous production of the same clones or recurrent activation of B cells generating new clones. To examine the stability of the human antibody repertoire, we monitored the concentrations of the most abundant IgG1 clones in plasma samples of 11 healthy donors at nine sampling points over a year. During this year, each donor received three doses of a COVID-19 vaccine. Notwithstanding these vaccinations, the concentrations of the most abundant IgG1 clones remained constant. Given the 2- to 3-week half-life of IgG1 molecules in blood, our data suggest that these clones are associated with long-term immunity and do not undergo somatic hypermutation which would imply short-lived plasma cells. Overall, our data suggest that most of the abundant IgG1 clones in plasma are persistently produced by long-lived plasma cells.

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.

The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.

Abstract Background Immunoglobulin (Ig) glycosylation modulates the immune response and plays a critical role in ageing and diseases. Studies have mainly focused on IgG glycosylation, and little is known about the genetics and epidemiology of IgA glycosylation. Methods We generated, using a novel liquid chromatography-mass spectrometry method, the first large-scale IgA glycomics dataset in serum from 2423 twins, encompassing 71 N- and O-glycan species. Results We showed that, despite the lack of a direct genetic template, glycosylation is highly heritable, and that glycopeptide structures are sex-specific, and undergo substantial changes with ageing. We observe extensive correlations between the IgA and IgG glycomes, and, exploiting the twin design, show that they are predominantly influenced by shared genetic factors. A genome-wide association study identified eight loci associated with both the IgA and IgG glycomes (ST6GAL1, ELL2, B4GALT1, ABCF2, TMEM121, SLC38A10, SMARCB1, and MGAT3) and two novel loci specifically modulating IgA O-glycosylation (C1GALT1 and ST3GAL1). Validation of our findings in an independent cohort of 320 individuals from Qatar showed that the underlying genetic architecture is conserved across ancestries. Conclusions Our study delineates the genetic landscape of IgA glycosylation and provides novel potential functional links with the aetiology of complex immune diseases, including genetic factors involved in IgA nephropathy risk.

IntroductionUpon vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humans will start to produce antibodies targeting virus specific antigens that will end up in circulation. In lactating women such antibodies will also end up in breastmilk, primarily in the form of secretory immunoglobulin A1 (SIgA1), the most abundant immunoglobulin (Ig) in human milk. Here we set out to investigate the SIgA1 clonal repertoire response to repeated SARS-CoV-2 vaccination, using a LC–MS fragment antigen-binding (Fab) clonal profiling approach.MethodsWe analyzed the breastmilk of six donors from a larger cohort of 109 lactating mothers who received one of three commonly used SARS-CoV-2 vaccines. We quantitatively monitored the SIgA1 Fab clonal profile over 16 timepoints, from just prior to the first vaccination until 15 days after the second vaccination.ResultsIn all donors, we detected a population of 89–191 vaccine induced clones. These populations were unique to each donor and heterogeneous with respect to individual clonal concentrations, total clonal titer, and population size. The vaccine induced clones were dominated by persistent clones (68%) which came up after the first vaccination and were retained or reoccurred after the second vaccination. However, we also observe transient SIgA1 clones (16%) which dissipated before the second vaccination, and vaccine induced clones which uniquely emerged only after the second vaccination (16%). These distinct populations were observed in all analyzed donors, regardless of the administered vaccine.DiscussionOur findings suggest that while individual donors have highly unique human milk SIgA1 clonal profiles and a highly personalized SIgA1 response to SARS-CoV-2 vaccination, there are also commonalities in vaccine induced responses.

AbstractImmunoglobulin (Ig) glycosylation modulates the immune response, and plays a critical role in ageing and diseases. Studies have mainly focused on IgG glycosylation, and little is known about the genetics and epidemiology of IgA glycosylation. Here, we generated, using a novel LC-MS method, the first large-scale IgA glycomics dataset in serum from 2,423 twins, encompassing 71N-andO-glycan species. We showed that, despite the lack of a direct genetic template, glycosylation is highly heritable, and that glycopeptide structures are sex-specific, and undergo substantial changes with ageing. We observe extensive correlations between the IgA and IgG glycomes, and, exploiting the twin design, show that they are predominantly influenced by shared genetic factors. A genome-wide association study identified eight loci associated with both the IgA and IgG glycomes (ST6GAL1,ELL2,B4GALT1,ABCF2,TMEM121,SLC38A10,SMARCB1,MGAT3), and two novel loci specifically modulating IgAO-glycosylation (C1GALT1andST3GAL1). Validation of our findings in an independent cohort of 320 individuals from Qatar showed that the underlying genetic architecture is conserved across ethnicities. Our study delineates the genetic landscape of IgA glycosylation and provides novel potential functional links with the aetiology of complex immune diseases, including genetic factors involved in IgA nephropathy risk.

Here, we employed a variety of mass spectrometry (MS)-based approaches, both (glyco)peptide-centric and protein-centric, to resolve the complex glycoproteoform landscape of recombinant IgA1 produced in HEK293 cells. These key immunoglobulins harbor several N- and O-glycosylation sites, making them considerably more heterogeneous than their IgG counterparts. We provide quantitative data on the occupancy and glycan composition for each IgA1 glycosylation site. Combining all data, we revealed that IgA1 molecules consist of at least three distinct populations with varying N-glycosylation site occupancies at the C-terminal tailpiece, namely, one with both glycosylation sites occupied, another with both glycosylation sites unoccupied, and a third asymmetric population with one glycosylation site occupied and the other unoccupied, challenging the prevailing acceptance that IgA1 N-glycosylation is symmetrical. This finding is significant, given that the tailpiece is involved in interactions with the J-chain and the Polymeric Immunoglobulin Receptor, and in general as antibody glycosylation is a quality attribute that needs to be carefully monitored, as the presence and nature of these modifications can affect the antibody's efficacy, lifetime, stability, and binding and/or neutralizing capacities. Optimizing strategies to produce recombinant IgA1 requires efficient and specific quality control analytical strategies, as presented here, which is essential for therapeutic IgA1-based antibody development. We expect that the integrated MS-based strategy presented here may be beneficial to comprehensively characterize the glycoproteoform profiles of IgA1-based therapeutics, thereby improving their production and optimization processes and facilitating the pathway to bring more IgA1-based therapeutics into clinical applications.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Serum proteomics has matured and is now able to monitor hundreds of proteins quantitatively in large cohorts of patients. However, the fine characteristics of some of the most dominant proteins in serum, the immunoglobulins, are in these studies often ignored, due to their vast, and highly personalized, diversity in sequences. Here, we focus exclusively on these personalized features in the serum proteome and distinctively chose to study individual samples from a low diversity population: elderly donors infected by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). By using mass spectrometry-based methods, immunoglobulin IgG1 and IgA1 clonal repertoires were monitored quantitatively and longitudinally in more than 50 individual serum samples obtained from 17 Corona virus disease 2019 patients admitted to intensive care units. These clonal profiles were used to examine how each patient reacted to a severe SARS-CoV-2 infection. All 17 donors revealed unique polyclonal repertoires and substantial changes over time, with several new clones appearing following the infection, in a few cases leading to a few, very high, abundant clones dominating their repertoire. Several of these clones were de novo sequenced through combinations of top-down, middle-down, and bottom-up proteomics approaches. This revealed sequence features in line with sequences deposited in the SARS-CoV-specific antibody database. In other patients, the serological Ig profiles revealed the treatment with tocilizumab, that subsequently dominated their serological IgG1 repertoire. Tocilizumab clearance could be monitored, and a half-life of approximately 6 days was established. Overall, our longitudinal monitoring of IgG1 and IgA1 repertoires of individual donors reveals that antibody responses are highly personalized traits of each patient, affected by the disease and the chosen clinical treatment. The impact of these observations argues for a more personalized and longitudinal approach in patients' diagnostics, both in serum proteomics as well as in monitoring immune responses.

Abs can be glycosylated in both their Fc and Fab regions with marked effects on Ab function and binding. High levels of IgG Fab glycosylation are associated with malignant and autoimmune conditions, exemplified by rheumatoid arthritis and highly Fab-glycosylated (∼90%) anti-citrullinated protein Abs (ACPAs). Important properties of IgG, such as long half-life and placental transport, are facilitated by the human neonatal Fc receptor (hFcRn). Although it is known that glycosylation of Abs can affect binding to Fc receptors, little is known on the impact of IgG Fab glycosylation on hFcRn binding and transplacental transport. Therefore, we analyzed the interaction between hFcRn and IgG with and without Fab glycans in vitro with various methods as well as in vivo by studying placental transfer of Fab-glycosylated Abs from mothers to newborns. No effect of Fab glycosylation on IgG binding to hFcRn was found by surface plasmon resonance and hFcRn affinity chromatography. In contrast, studies in a cell membrane context revealed that Fab glycans negatively impacted IgG-hFcRn interaction. In line with this, we found that Fab-glycosylated IgGs were transported ∼20% less efficiently across the placenta. This appeared to be a general phenomenon, observed for ACPAs, non-ACPAs, as well as total IgG in rheumatoid arthritis patients and healthy controls. Our results suggest that, in a cellular context, Fab glycans inhibit IgG-hFcRn interaction and thus negatively affect the transplacental transfer of IgG. As Fab-glycosylated Abs are frequently associated with autoimmune and malignant disorders and may be potentially harmful, this might encompass a regulatory mechanism, limiting the half-life and transport of such Abs.

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma.

Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder characterized by the presence of a predominant monoclonal antibody (i.e., M-protein) in serum, without clinical symptoms. Here we present a case study in which we detect MGUS by liquid-chromatography coupled with mass spectrometry (LC-MS) profiling of IgG1 in human serum. We detected a Fab-glycosylated M-protein and determined the full heavy and light chain sequences by bottom-up proteomics techniques using multiple proteases, further validated by top-down LC-MS. Moreover, the composition and location of the Fab-glycan could be determined in CDR1 of the heavy chain. The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and multiple myeloma, with fine molecular detail. The ability to detect monoclonal gammopathies and determine M-protein sequences straight from blood samples by mass spectrometry provides new opportunities to understand the molecular mechanisms of such diseases.

Using a recently introduced efficient mass spectrometry-based approach we monitored in molecular detail the IgG1 clonal responses in individual donorsindividual donors’ IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.