Identification of a Tetanus Toxin Specific Epitope in Single Amino Acid Resolution.

Vaccinations are among the most potent tools to fight infectious diseases. However, cross-reactions are an ongoing problem and there is an urgent need to fully understand the mechanisms of the immune response. For the development of a methodological workflow, the linear epitopes in the immune response to the tetanus toxin is investigated in sera of 19 vaccinated Europeans applying epitope mapping with peptide arrays. The most prominent epitope, appearing in nine different sera (923 IHLVNNESSEVIVHK937 ), is investigated in a substitution analysis to identify the amino acids that are crucial for the binding of the corresponding antibody species - the antibody fingerprint. The antibody fingerprints of different individuals are compared and found to be strongly conserved (929 ExxEVIVxK937 ), which is astonishing considering the randomness of their development. Additionally, the corresponding antibody species is isolated from one serum with batch chromatography using the amino acid sequence of the identified epitope and the tetanus specificity of the isolated antibody is verified by ELISA. Studying antibody fingerprints with peptide arrays should be transferable to any kind of humoral immune response toward protein antigens. Furthermore, antibody fingerprints have shown to be highly disease-specific and, therefore, can be employed as reliable biomarkers enabling the study of cross-reacting antigens.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays.

Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.