Antonia W. Godehardt, Ralf R. Tönjes

Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Division of Medical Biotechnology, D-63225 Langen, Germany

Xenotransplantation of porcine organs, tissue and cells inherit the general risk for xenozoonotic infections that can not be ruled out completely. Despite their compelling potential xenotransplants have to be considered as potentially contaminated, non sterile products (i) because of limited microbial examination methods for living primary cell cultures (see European Pharmacopoeia) as well as (ii) the presence of inherited porcine endogenous retroviruses, PERV-A/-B/-C/-A/C, that are highly polymorphic proviral loci in the pig genome. As a consequence, transmission of microorganisms and viruses has to be restricted by generating donor pigs raised under designated-free or qualified pathogen-free or specific pathogen free conditions. Furthermore, sterile manufacture of the product and the development of adequate screening methods, e.g. microarray technology, that provide the required information on the microbial status in time need to be performed without persuasion of the source material.

The detection of PERVs in donor animal tissues and first xenotransplantation recipients hereby includes cryopreserved samples of peripheral blood mononuclear cells (PBMC) and allows a retrospective testing for antibodies and viruses in blood and in PBMC.

The assays base first on direct PCR screening to detect either provirus DNA or quantitative real-time reverse transcriptase PCR analyzing the release of packaged RNA particles in the cell supernatant or quantifying the PERV reverse transcriptase activity in the supernatant. Second, indirect screening bases on the humoral response thereby screening for PERV specific antibodies via ELISA and Western blot analyses or immunofluorescence assays. In vitro assays with cell supernatants containing competent, packaged virus particles and co-cultivation experiments using PBMCs from the patient and highly susceptible human cells provide additional information on virus infectious potential.

However, to face the quality questions raised and to provide an in vivo insight, thereby monitoring tissues viability as well as its microbial/viral status the assays were extended by generation of a spotted 60K DNA microarray displaying the whole Sus scrofa genome currently published at NCBI. The microarray was specified amongst others for German Landrace and Göttingen Minipig by hybridizing complex-RNA samples and chromosomal DNA. Functional PERV sequences for PERV-A/-B/-C prt/pol, PERV-A env, PERV-B env and PERV-C env as well as several human transgenes were included in order to monitor the transcriptional status of fresh porcine tissue that is intended as xenotransplant in order to generate tissue products that reveal comparable quality standards.

In addition to the results gained, a second microarray will be generated in extension to the first array for simultaneous, species specific identification of human and porcine, pathogenic bacteria, viruses, parasitic protozoa and fungi.