Bacteria reveal targets of immune responses quick and direct identification of antigens

The Epstein-Barr virus (EBV) is a widespread herpes virus that infects more than 90 per cent of the worldwide human population.

After primary infection, EBV persists for life in the infected human host in a state of latency. In healthy individuals, the immune system is able to control latent EBV infection.

If, however, the immune system is suppressed, e.g. by AIDS or following transplantation, EBV can reactivate and cause the development of malignant tumours. These EBV-positive tumours can be treated by the infusion of immune cells (T cells) that are specific for components of the virus (antigens). An elegant and quick method developed by a Clinical Cooperation Group of the GSF – Research Center for Environment and Health and the Children’s Hospital of the University of Technology, Munich (TUM), helps to identify such targets of attack. The trick: bacteria are first genetically modified to produce all EBV proteins. With the help of T cells which are known to be EBV-specific, a test is conducted to identify those EBV proteins that act as antigens.

EBV-specific, cytotoxic and helper T cells have been successfully used to treat EBV-associated tumours, but it remained unknown which antigens the T cells attack. Therefore, T cells for immunotherapy had to be generated using laborious and tedious methods. "So far, the generation of EBV-specific T cells has taken two to three months, which is too long for many immunosuppressed patients with EBV-associated complications. Apart from that, the production is too complicated and too expensive as to perform it for each patient prophylactically,” says PD Dr. Uta Behrends, the head of the Clinical Cooperation Group Paediatric Tumour Immunology, “if the relevant EBV-antigens were known, the treatment could ideally be available within a few days.”

To this end, the scientists developed a detection method by which helper T cell antigens can be identified directly: DANI (Direct Antigen Identification). The principle: fragments of the EBV genome are introduced into E. coli bacteria, and the viral DNA fragments are translated into viral proteins by the bacteria. This works particularly well, if the introduced DNA fragments are small and only pieces and not the whole viral proteins are produced. Because such small protein fragments are unstable in bacteria, they must be coupled to a carrier protein to protect them from degradation. In the DANI method, the Chloramphenicol acetyltransferase (CAT) is used as carrier protein because CAT offers two advantages: first, protein fragments fused to CAT are expressed at high levels, even if they are derived from proteins that are toxic to bacteria, and second, by adding the antibiotic chloramphenicol to bacterial cultures, only those bacteria survive that form a functional fusion protein: only bacteria expressing CAT can inactivate chloramphenicol and form colonies - since this applies to only about one tenth of the bacteria, the number of colonies to screen is reduced considerably.

The chloramphenicol-resistant bacteria are “fed” to antigen-presenting cells, which “eat” the bacteria and digest bacterial proteins -including the EBV-CAT fusion proteins- into small protein fragments (peptides) and present these on HLA receptors at the cell surface. In a next step, EBV-specific T cells are added to these antigen-presenting cells – if this results in an immune reaction, the breakdown products have been identified as antigens. Using this method, the Clinical Cooperation Group identified, e.g. the EBV proteins BALF4 and BNRF1 as target structures of T helper cells. Both proteins are expressed during the lytic phase of infection, when new viral particles are assembled and viral progeny is released from infected cells.

In addition to the identification of EBV antigens, the DANI method may also be applied to identify tumour- and transplantation-associated antigens, or autoantigens, which may facilitate the development of immunotherapies for these diseases. “For example leukaemia relapses after bone marrow transplantation might be prevented with T cells specific for transplantation-associated antigens” hopes Behrends, whose research group at the GSF and TUM has started searching for tumour- and transplantation-associated antigens with the DANI method.