Ligand Development

Coronaviruses have accompanied mammals for a very long time. At least four different endemic corona viruses are circulating in humans and have left an imprint on the immune system. Therefore, this project aimed not only at identifying epitopes recognized by antibodies after SARS-CoV-2 infection, but also, the endemic relatives. By applying the epitope fingerprinting technology developed in this working group, even the small differences in amino acid sequences of highly similar viral proteins can be observed. The analysis identified SARS-CoV-2-specific epitopes, and also epitopes recognized by very similar antibodies in three patient groups: patients with respiratory infections before 2020, COVID-19 patients and vaccinated patients. A large number of peptide epitopes were printed as arrays on slides, and tested using more than 1000 serum samples. The quantity of serum antibodies bound to peptide spots could be recorded and analysed easily with the help of fluorescence labelling, followed by scanning in the laser scanner, and using a new, robust automated analysis of the images generated. This new image analysis system was developed in the framework of the project. It was thanks to this development that such a large number of measurements could be analysed quickly and reliably.

By correlating the results from the previously mentioned three patient groups, large varieties of antibodies against the S-protein and RNA-polymerase were identified, originating from previous infections with endemic strains. Some antibodies circulate in serum already before the vaccine or immediately after SARS-CoV-2 infection. Surprisingly, some cross-reactive antibodies recognize also epitopes in S-protein, with highly identical structure despite their distinct amino acid sequences (Fig. 1).

In future, these and other epitopes can help to track specific individual antibodies rather than tracking antibodies on a protein globally. This would permit tracking of the individually very diverse immune responses to COVID-19 and other vaccines. 

At present, serological diagnostics for COVID-19 are only offered based on proteins. In addition, to unexpected false positives, clinical diagnostics specifically report problems with previous infections with related Corona viruses. This is because the recognition sites of the patients’ antibodies (epitopes) are only partly specific to SARS-CoV-2, while others are found in many related Corona viruses.

Therefore, in future, serological tests will also have to be developed on the basis of defined epitopes of SARS-CoV-2 or other Corona viruses which permit both simple and highly individualized diagnostics with the help of different specific and ubiquitous epitopes. The Ligand Development Unit at Fraunhofer IZI has comprehensive experience in identifying epitopes directly from serums. It is already evident that SARS-CoV-2 infections lead to a strongly personalized immune response which is shaped by previous Corona infections.

However, in all diagnostic activities, the serums obtained from infected patients are pre-treated to inactivate the virus before the serums can be used in testing. In the context of CoV-tot, it was demonstrated that different methods for virus inactivation have a very different influence on different antibodies in the sera. This can lead to very different results in serological tests in individual cases. Furthermore, the studies have shown that a slight denaturation of the sample seems to remove the blocking of individual antibodies by unknown serum components. Thus, better results can be obtained. 

Two methods of viral inactivation have been identified as suitable, firstly heating to 56 degrees Celsius for ten minutes for liquid samples and secondly treatment with 70 % ethanol when antibodies are immobilized on protein A.

The knowledge gained in CoV-tot is a starting point to explore differences in disease progression also in the context of clinical trials for drug treatment of COVID-19.

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Klinikum St. Georg, Leipzig, Germany  |  www.sanktgeorg.de

Support
This work was supported by the Fraunhofer InternaI Programs under Grant No. Anti-Corona 131-600034.

Currently immunodiagnostics for diseases are usually based on proteins or extracts, which are directly obtained from the pathogenic organism or produced with biotechnological methods. The disadvantage of this approach is that variants, as they are for example commonly observed for the influenza virus, are difficult to distinguish. We have established protocols to exactly identify the antibody binding sites (epitopes) of patient antibodies, which are also directly applicable to sera. This allows a reliable identification of the pathogen, the causative antigen of allergies or many indications such as (auto)-immune or infectious disease as well as novel approaches for therapy and research.

Food allergies have been one focus of our research over many years. A steady increase of patients could be observed in the recent years. Skin prick tests diagnostics are only of limited use because many plant proteins are very similar in their architecture. Epitope-based diagnostics are most likely the only alternative to elaborate clinical investigations. These usually require venous blood collection, although only the provocation with the food is regarded as proof of an allergy, which has to be carried out under medical supervision in a clinic. An efficient diagnosis, appropriate treatment and adjustment of the food is therefore not available for many patients.

In an initial project we could show as an example for soy allergies that indeed only a few epitopes are sufficient for safely identifying sensitized persons as well as such with clinical symptoms. We are working to use these peptides in a simple test that could detect antibodies in a single drop of blood. Such a test would also be a model for tests on infectious diseases, vaccine efficacy or autoimmune diseases.

A particularly large project is now being funded by the Fraunhofer-Zukunftsstiftung, which is being followed nationally and internationally by allergologists with great interest. In cooperation with several other Fraunhofer Institutes and hospitals, the FoodAllergen project is working on a holistic approach to deal with food allergies. This also includes identifying allergens in foods and new processes of producing food ingredients with reduced allergenic potential. Meanwhile, the epitopes for a wide variety of plant allergens have been identified. An application in tests for patients is in preparation.

In a joint project with Fraunhofer IFAM (Bremen) we discovered an enzyme based reaction for covalent of peptide ligands to cell surface receptors. We initiated the Fraunhofer society sponsored project ZELLFIX to use a similar reaction to improve the attachment of cells to polymer surfaces. It is sometimes very difficult to achieve the attachment of cells to the surfaces of culture dishes. Usually polystyrene surfaces are treated with plasma which can result in toxic byproducts. With this new procedure we improve cell binding through peptides or proteins to the polymer surface or by direct binding of these proteins to the polymers just like to plasma activated surfaces. Within ZELLFIX we managed to obtain the expected results. Even local spots can be modified to enhance cell binding. Finally we have developed an enzymatic method that provides the same results as the plasma treatment of polymers without toxic side effects. This method is presently being tested with several partners from the industry and other Fraunhofer and research institutes. We have identified multiple applications in biological and medical areas and beyond.