Ligand Development


Development of glycosylated vaccine proteins

Lage der Epitope (rot) in einem Modell des E.coli YghJ Proteins
© Fraunhofer IZI
Position of the epitopes (red) in a model of the E.coli YghJ protein, which was able to be validated using peptide arrays.

Uropathogenic E. coli strains often cause serious urinary tract infections, especially in patients admitted to hospital for the treatment of other diseases. Besides the added strain on patient health, this also gives rise to substantial additional treatment and follow-up costs. 

The aim of this project is to develop a vaccine that strengthens the immune response to urinary tract infections, especially in hospital patients. The idea here is for the immune response to specifically target the bacterial protein YghJ, which is essential for the colonization of the urinary tract.

Researchers from the Danish company GlyProVac LLC. have discovered that bacteria such as E. coli modify a large proportion of their proteins using sugar molecules (glycosylation). These modifications have a significant bearing on the immune response and are therefore also of relevance to the development of protective vaccines. GlyProVac LLC. has developed ways of verifying and analyzing these modifications. As part of the project, special host strains will now be developed to manufacture recombinant proteins that are very similar to the naturally occurring YghJ protein but induce an enhanced immune response.

In order to study the immune response to glycosylated and non-glycosylated YghJ vaccine proteins in detail and compare it with the immune response to the natural antigen, the antibodies produced by the immune system must be precisely analyzed. The methods available to date, however, have proven inadequate here. 

Researchers from Fraunhofer IZI and epitopic GmbH have thus developed a procedure for the quick and precise identification and analysis of epitopes, i.e. the molecular structures which are recognized and bound by antibodies. Using the procedure, more than 20 epitopes of the YghJ protein were able to be identified and can be drawn upon when evaluating the vaccine candidates (see figure). Further improving the methods should also allow structures to be found that target glycosylation itself. 

Comparisons between the animal model and patient serums could already confirm that the developed vaccine candidates trigger an immune response that resembles the natural reaction. The verification procedures developed within the scope of this project will also bear significance for future clinical development.

GlyProVac LLC.; epitopic GmbH; Odense University Hospital; Klinikum St. Georg Leipzig

Grant number 01QE2109B
Logo Eurostars
Logo EU Eureka

Biosensors for the detection of infecting agents

Eingebetteter Messchip mit BDD-(Bor-dotierter Diamant)-­Oberfläche
© Fraunhofer IZM
Embedded measuring chip with a BDD (boron-doped diamond) surface.

The corona pandemic made clear just how important it is to have robust technologies in place for the rapid and accurate analysis of disease agents and antibodies in order to monitor infection outbreaks and immunity among larger population groups. This project aims to develop an immunodiagnostic technology platform that can be flexibly adapted to dynamic pandemic activity.

Electrodes made from boron-doped diamond (BDD) are key to this development; they are combined with epitopes, i.e. structures that exist in the pathogen and are recognized by the antibody, to create sensitive and efficient biosensors. 

The specific detection of SARS-CoV-2 was initially focused on as a demo application. Fraunhofer IZI is using its expertise to help identify and develop peptide-based epitopes here, which will be taken as a basis for developing sensitive and specific detection systems. Epitopes will be utilized that are already available from projects involving hundreds of patient serums. Serums taken from COVID-19 patients infected with omicron were also used in the project. At the same time, with an eye to long COVID, patient serums and data were analyzed for potential epitopes of autoantigens against proteins produced naturally in the body, which are said to play an important role in the development of long COVID.

Following a modular concept, the platform should be flexibly and quickly adaptable to different disease agents or immunologically relevant questions. The project aims to make devices available for point-of-care diagnostics, for quick and cost-efficient monitoring of infectious diseases and for determining the immune status of vaccinated or recovered individuals within just a few weeks of the outbreak of an infection in the future.

Fraunhofer USA Center Midwest CMW, Coatings and Diamond Divison; Fraunhofer Institute for Reliability and Microintegration IZM

EpiCoV2020 –SARS-CoV-2 specific serological diagnostics based on epitopes

Abbildung 1 zeigt links die überlagerten Strukturen der S-Proteine von SARS-CoV-2 und dem endemischen Coronavirus 229E. Zwei praktisch identische Bereiche, die kreuzreagierende Antikörper erzeugen sind grün und schwarz gekennzeichnet. Die dazugehörenden Spots im Peptid­array rechts entsprechend umrandet. Das Bild zeigt die Ergebnisse vor und nach Impfung. In diesem Fall liegt wahrscheinlich eine bestehende Immunantwort auf Coronavirus 229E vor und nach der Impfung entwickeln sich SARS-CoV-2 spezifische und auch kreuzreaktive Antikörper.
Abbildung 1 zeigt links die überlagerten Strukturen der S-Proteine von SARS-CoV-2 und dem endemischen Coronavirus 229E. Zwei praktisch identische Bereiche, die kreuzreagierende Antikörper erzeugen sind grün und schwarz gekennzeichnet. Die dazugehörenden Spots im Peptid­array rechts entsprechend umrandet. Das Bild zeigt die Ergebnisse vor und nach Impfung. In diesem Fall liegt wahrscheinlich eine bestehende Immunantwort auf Coronavirus 229E vor und nach der Impfung entwickeln sich SARS-CoV-2 spezifische und auch kreuzreaktive Antikörper.

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. 

CoV-tot – Examination of the influence of virus inactivation on the epitope spectrum in (COVID-19) serums

Blut in Tubes
© Fraunhofer IZI
Das Signal verschiedener Antikörper im Peptid­array wird durch die Vorbehandlung individuell stark reduziert. Teilweise sind die Antikörper nicht mehr nachweisbar.
The signal of antibodies against different epitopes in the peptide array is individually and significantly reduced through pretreatment. Some of the antibodies can no longer be detected and are probably destroyed by the application of an inactivation method.

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.

Klinikum St. Georg, Leipzig, Germany  |

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

Mapping of allergen epitopes in sera

© S.Piyaset – Fotolia

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.

Epitope / mimotope mapping

Peptide Phage Display libraries of the size available at Fraunhofer IZI allow a rapid and reliable identification of epitopes and mimotopes of monoclonal as well as polyclonal antibodies. The identified peptides allow a rapid identification of the exact binding site on the antigen. They can also be used in serological assays and for the purification of recombinant antibodies or specific antibodies from polyclonal mixtures.

This rapid routine work is very helpful in the early stages of antibody development as well as for the precise understanding of the spectrum of peptides bound by the individual antibody.

ZellFix – Enzymatic treatment replacing plasma: Biocompatible polymer surfaces

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.