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Yucatan SLA D/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro , the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

INTRODUCTION: Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. HYPOTHESIS/GAP STATEMENT: In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk.AIM: We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/−1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. METHODOLOGY: Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. RESULTS: Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%–96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day 7. CONCLUSION: The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

Background The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. Methods Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. Results This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. Conclusions In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future.

Background The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. Results The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. Conclusions The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.

BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103 copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Background Porcine induced pluripotent stem cells (piPSCs) offer an alternative strategy in xenotransplantation (XTx). As human endogenous retroviruses (HERV), particularly HERV‐K, are highly expressed in natural human stem cells, we compared the expression of porcine endogenous retroviruses (PERV) and retrotransposon LINE‐1 (L1) open reading frames 1 and 2 (pORF1 and pORF2) in different piPSC‐like cell lines with their progenitors (porcine fetal fibroblasts, pFF). Methods Cells reprogrammed via Sleeping Beauty‐transposed transcription factors were cultured and analyzed on a custom‐designed microarray representing the reference pig genome. Data were complemented by qRT‐PCR and reverse transcriptase (RT) assay. Results The expression profiles revealed that 8515 of 26 967 targets were differentially expressed. A total of 4443 targets showed log2 expression ratio >1, and 4072 targets showed log2 expression ratio less than −1 with 0.05 P‐value threshold. Approximately ten percent of the targets showed highly significant expression ratios with log2 ≥4 or ≤−4. Besides this general switch in cellular gene expression that was accompanied by an altered morphology, expression of both PERV and L1 pORF1/pORF2 was significantly enhanced. piPSC‐like cells revealed a 10‐fold to 100‐fold higher transcription of the viral PERV‐A and PERV‐B envelope genes (env), viral protease/polymerase (prt/pol), and L1 elements. No functional retrovirus could be detected under these conditions. Conclusion Epigenetic reprogramming has functional impact on retrotransposons. Thus, the induction of pig‐derived pluripotent cells influences their PERV expression profile. Data emphasize the necessity to focus on animals, which show non‐functional endogenous viral background to ensure virological safety.

Angesichts der bestehenden Knappheit an humanen Spenderorganen und -geweben kann die Entwicklung xenogener Zelltherapeutika (xZT) eine adaquate Behandlungsalternative sein. Insbesondere porcine Pankreasinseln und -inselzellen haben bereits zum jetzigen Zeitpunkt Eingang in die experimentelle Therapie von Typ 1 Diabetes mellitus (T1DM)-Patienten gefunden. Dabei stellen xZT sowohl aus medizinischer als auch aus ethischer und regulatorischer Sicht eine Herausforderung dar. So ist infolge der speziesubergreifenden Transplantation (Xenotransplantation) neben dem Risiko der immunologischen Abstosung auch das Infektionsrisiko durch Ubertragung von mikrobiellen und viralen Erregern zu berucksichtigen. Letzteres ist bidirektional und betrifft sowohl die Transmission vom Transplantat auf den Empfanger als auch die Transmission vom Empfanger auf das Transplantat. Die Uberschreitung der Speziesbarriere erfordert eine kritische Nutzen-Risiko-Bewertung und nach Abschluss der Behandlung eine sorgfaltige und langfristige Uberwachung der Patienten. Die internationalen rechtlichen und regulatorischen Anforderungen an xZT basieren u. a. auf den von der Weltgesundheitsorganisation (WHO) 2008 im Changsha Kommunique formulierten Kriterien, die in Europa nach Inkraftsetzen der EU-Verordnung uber Arzneimittel fur neuartige Therapien (ATMP) im Leitfaden fur xenogene Zelltherapeutika seitens der Europaischen Arzneimittelbehorde EMA mit abgebildet sind.

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.

Am 1. August 2007 trat in Deutschland das Gewebegesetz in Kraft, welches unter anderem Regelungen hinsichtlich der Qualitat und Sicherheit von Gewebezubereitungen (GWZ) trifft. Klassische GWZ sind Arzneimittel fur die Anwendung am Menschen, a) die menschliche Gewebe und Zellen enthalten oder ausgehend von diesen hergestellt werden, b) deren wesentliche Be- oder Verarbeitungsverfahren in der EU hinreichend bekannt bzw. neu, aber mit bekannten Verfahren vergleichbar sind, und c) deren Wirkungen und Nebenwirkungen aus dem wissenschaftlichen Erkenntnismaterial ersichtlich sind. Dazu gehoren z.B. Herzklappen, Corneae, muskuloskelettale GWZ, Gefase oder Stammzellen aus Knochenmark. Wenn bei einer GWZ die oben genannten Voraussetzungen erfullt sind und diese nicht mit ionisierenden Strahlen behandelt wird, ist fur das Inverkehrbringen eine Genehmigung gemas § 21a Abs. 1 Arzneimittelgesetz (AMG) durch das Paul-Ehrlich-Institut erforderlich. Zur Erleichterung der Antragstellung fur die Genehmigung stehen Formulare auf der Internetseite des Paul-Ehrlich-Instituts zur Verfugung. Schwerwiegende unerwunschte Reaktionen und schwerwiegende Zwischenfalle gemas § 63c AMG mussen von Gewebeeinrichtungen unverzuglich an die zustandige Bundesbehorde gemeldet werden. Des Weiteren besteht fur Gewebeeinrichtungen eine Meldepflicht gemas § 8d Abs. 3 Transplantationsgesetz (TPG) zur Erfassung der verarbeiteten Gewebe, die in Form eines jahrlichen Berichts bis zum 1. Marz des folgenden Jahres an das Paul-Ehrlich-Institut erfolgt.

One of the major problems associated with the development of a vaccine against Streptococcus pyogenes is the ability of this pathogen to escape recognition by antibodies directed against conserved surface-associated determinants and to establish infection in the setting of an acquired immune response. Identification of the mechanism(s) used by S. pyogenes to avoid recognition by antigen-specific antibodies and escape killing in blood was the focus of this study. We showed here that S. pyogenes was capable of surviving in human blood containing high levels of antibodies directed against the G-related alpha2-macroglobulin-binding protein GRAB, a highly conserved bacterial surface protein. S. pyogenes upregulated the hyaluronic acid capsule production during incubation with human blood, suggesting that the capsule may structurally minimize antibody access to protein GRAB. This hypothesis was confirmed by the ability of anti-GRAB antibodies to promote opsonophagocytosis of a capsule-deficient mutant of S. pyogenes but not of the encapsulated wild-type strain. Capsule upregulation and protection of S. pyogenes from opsonophagocytosis in the presence of anti-GRAB antibodies was also observed in a murine model of streptococcal infection. Thus, masking of surface immunogenic determinants by the hyaluronic acid capsule may constitute a novel mechanism of S. pyogenes for evasion of antigen-specific antibodies.

In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42--Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.

Pig to human xenotransplantation represents an ambitious venture that requires, besides evasion of rejection mechanisms and physiological incompatibilities, the generation of pathogen-free pigs as donors for well characterized xenografts to warrant medicinal products that do comply with statutory regulatory demands [1–3]. The publication of a high quality draft sequence for the pig genome (Sus scrofa) and a series of accompanying reports for the first time offered the feasibility of whole genome expression profiling of porcine tissues and cells [4–8]. The SFB TR CRC 127 project Z2 “Microbiological Safety including Virological Safety” is based on microbial profile analysis of porcine tissues in order to prevent zoonotic events, including infection by porcine endogenous retroviruses (PERV) [9]. The project comprises the detection and characterization of potential pathogens as well as the investigation of the microbial influence on the transcriptional status of tissues and cells. Hence, specific expression patterns, e.g. up-regulation of antiviral host factors or cell cycle/apoptotic regulators, may also provide information on ongoing or precedent events that may have impact on tissues/cells quality and therefore its suitability as xenografts. We use microarray technology for monitoring viability of tissues and cells as well as their microbial/viral status. An Agilent based, 60K DNA microarray representing 25,415 different genes of the recently published Sus scrofa genome (NCBI Sus scrofa 10.2-assembly) was generated [10]. The microarray was specified for German Landrace and Gottingen Minipig, amongst other pig species, by hybridizing complex RNA samples generated from five different pig organs and blood as well as chromosomal porcine DNA to highlight non expressed genes. Four Diagnostic PERV sequences for pro/pol (all classes of PERV), env (to differentiate between PERV-A, -B and -C) as well as 15 human transgenes such as CD59 (human complement regulatory protein), DAF (Decay accelerating factor or CD55), human A20 (hA20) and others were included. In total, the microarray displays 25,434 genes each represented by up to three different 60-mer oligonucleotides. To reveal functionality of the microarray the transcriptional status of ST-IOWA cells freshly infected with molecularly cloned virus PERV-C (1312) [11] was monitored. Total mRNA levels at day 7, 28 and 56 post infection were compared with naive uninfected cells. All samples were tested in triplicates and the relative signal intensity of hybridized probes was compared. Special attention was given to antiviral host factors such as APOBEC and tetherin of which involvement as antiviral factors on PERV expression has been demonstrated [12–14]. Constitutively expressed housekeeping genes, i.e. porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin and cyclophilin A, respectively, were used as controls [15, 16]. The presented microarray supports the safety and quality by monitoring the transcriptional status of xenotransplants. References [1] EMEA/CHMP/CPWP/83508/2009. Guideline on Xenogeneic Cell-Based Medicinal Products. [2] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). February 2002. Draft Guidance for Industry: Precautionary Measures to Reduce the Possible Risk of Transmission of Zoonoses by Blood and Blood Products from Xenotransplantation Product Recipients and Their Intimate Contacts. [3] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). April 2003. Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans. [4] Groenen MA, et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 2012; 491(7424): 393–398. [5] Li Y, Mei S, Zhang X, et al. Identification of genome-wide copy number variations among diverse pig breeds by array CGH.BMC Genomics 2012; 13:725. doi: 10.1186/1471-2164-13-725. [6] Servin B, Faraut T, Iannuccelli N, Zelenika D, Milan D. High-resolution autosomal radiation hybrid maps of the pig genome and their contribution to the genome sequence assembly. BMC Genomics 2012; 13:585. doi: 10.1186/1471-2164-13-585. [7] Nguyen DT, Lee K, Choi H, et al. The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome. BMC Genomics 2012; 13:584. doi: 10.1186/1471-2164-13-584. [8] Uenishi H, Morozumi T, Toki D et al. Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence. BMC Genomics 2012; 13:581. doi: 10.1186/1471-2164-13-581. [9] Denner J, Tonjes RR. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin Microbiol Rev. 2012; 25(2): 318–743. [10] Agilent's Microarray Platform. How High-Fidelity DNA Synthesis Maximizes the Dynamic Range of Gene Expression Measurements. Library – Application Note. 2013; Publication Part Number: 5989-9159EN [11] Preuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006; 80(20):10258–61. [12] Dorrschuck E, Fischer N, Bravo IG, et al. Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases. J Virol. 2011; 85(8):3842–57. [13] Dorrschuck E, Munk C, Tonjes RR. APOBEC3 proteins and porcine endogenous retroviruses. Transplant Proc. 2008; 40(4):959–61. [14] Mattiuzzo G, Ivol S, Takeuchi Y. Regulation of porcine endogenous retrovirus release by porcine and human tetherins. J. Virol. 2010; 84: 2618 –2622. [15] Mcculloch RS, S. Ashwell M, O'Nan AT, Mente PL. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage. J Anim Sci Biotechnol. 2012; 3(1):36. [16] Nygard AB, Jorgensen CB, Cirera S, Fredholm M. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol. 2007; 8:67.