A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain.

Krüppel-associated box (KRAB) zinc finger proteins are a large class of tetrapod transcription factors that usually exert transcriptional repression through recruitment of TRIM28/KAP1. The evolutionary root of modern KRAB domains (mKRAB) can be traced back to an ancestral motif (aKRAB) that occurs even in invertebrates. Here, we first stratified three subgroups of aKRAB sequences from the animal kingdom (PRDM9, SSX and coelacanth KZNF families) and defined ancestral subdomains for KRAB-A and KRAB-B. Using human ZNF10 mKRAB-AB as blueprints for function, we then identified the necessary amino acid changes that transform the inactive aKRAB-A of human PRDM9 into an mKRAB domain capable of mediating silencing and complexing TRIM28/KAP1 in human cells when employed as a hybrid with ZNF10-B. Full gain of function required replacement of residues KR by the conserved motif MLE (positionsA32-A34), which inserted an additional residue, and exchange of A9/S for F, A20/M for L, and A27/R for V. AlphaFold2 modelling documented an evolutionary conserved L-shaped body of two α-helices in all KRAB domains. It is transformed into a characteristic spatial arrangement typical for mKRAB-AB upon the amino acid replacements and in conjunction with a third helix supplied by mKRAB-B. Side-chains pointing outward from the core KRAB 3D structure may reveal a protein-protein interaction code enabling graded binding of TRIM28 to different KRAB domains. Our data provide basic insights into structure-function relationships and emulate transitions of KRAB during evolution.

DUF3669, a "domain of unknown function" within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression.

BACKGROUND: ZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the "domain of unknown function 3669" (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson's disease. RESULTS: Here we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669's impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved "MLE" sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein. CONCLUSION: DUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

A computational method for designing diverse linear epitopes including citrullinated peptides with desired binding affinities to intravenous immunoglobulin.

BACKGROUND: Understanding the interactions between antibodies and the linear epitopes that they recognize is an important task in the study of immunological diseases. We present a novel computational method for the design of linear epitopes of specified binding affinity to Intravenous Immunoglobulin (IVIg). RESULTS: We show that the method, called Pythia-design can accurately design peptides with both high-binding affinity and low binding affinity to IVIg. To show this, we experimentally constructed and tested the computationally constructed designs. We further show experimentally that these designed peptides are more accurate that those produced by a recent method for the same task. Pythia-design is based on combining random walks with an ensemble of probabilistic support vector machines (SVM) classifiers, and we show that it produces a diverse set of designed peptides, an important property to develop robust sets of candidates for construction. We show that by combining Pythia-design and the method of (PloS ONE 6(8):23616, 2011), we are able to produce an even more accurate collection of designed peptides. Analysis of the experimental validation of Pythia-design peptides indicates that binding of IVIg is favored by epitopes that contain trypthophan and cysteine. CONCLUSIONS: Our method, Pythia-design, is able to generate a diverse set of binding and non-binding peptides, and its designs have been experimentally shown to be accurate.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients.

Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g.MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (pvalues <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392-411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS.

Epitope predictions indicate the presence of two distinct types of epitope-antibody-reactivities determined by epitope profiling of intravenous immunoglobulins.

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples.

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.

Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis.

Auto-antibodies are implicated in the pathophysiology of various autoimmune diseases. High-density peptide microarrays incubated with human serum can detect antibody reactivities against thousands of peptides. This enables the identification of new auto-antigens and the determination of the parts of protein antigens (epitopes) that are recognized by antibody paratopes. We discuss the utility of peptide microarrays to investigate epitope-antibody-recognitions (EAR) from systemic sclerosis to multiple sclerosis. The technology can help to establish reliable diagnostic and prognostic biomarkers employing a combination of antigenic peptides. We describe the specifics of peptide microarray data and present bioinformatic methods for their analysis. Quality control, data pre-processing and the filtering of specific peptides are demonstrated on an example data set. Peptide microarrays representing 24 selected proteins by 3235 overlapping 15mer peptides were used to measure antibodies in serum of 10 patients with limited cutaneous systemic sclerosis (SSC) and 10 healthy blood donors. The data showed a sparse and skewed distribution, and we observed strong individual differences since many peptide sequences were bound by antibodies of only one serum sample. In the sera of the SSc patients, but not of the healthy controls, we found antibodies to three peptides MGPRRRSRKPEAPRR, TPTPGPSRRGPSLGA and GPSRRGPSLGASSHQ that share a similar sequence motif (GP-R/S-RR). These peptides map to two known linear epitopes at the N-terminus of centromere protein A (CENPA), demonstrating the utility of peptide microarrays. Presented experimental and bioinformatic approach can be applied in the same manner for multiple sclerosis research.