Safety of Xenotransplantation: Development of screening methods and testing for porcine viruses

Xenotransplantation using pig cells, tissues or organs might be a promising solution to overcome the shortage for organs suitable for allotransplantation. Because of several reasons, the pig is currently the favoured donor species. However, the use of porcine xenotransplants is associated with the risk of transmitting porcine viruses to the human xenotransplant recipient. Among them porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHVs), porcine circovirus 2 (PCV2) and hepatitis E virus (HEV) play a role. Some of them cause immunosuppression and a zoonotic potential of others has been supposed. Therefore the possibility of direct transmission of those viruses between pigs and humans might be possible. Strategies to avoid the transmission of those pathogens are currently of main importance to increase lifetime of the transplant and therefore to save many lives of people standing on the transplant waiting list. To select virus-free animals as putative donor pigs and to recognise transmission of pathogens to transplant recipients, sensitive detection methods are needed. In this study the prevalence and expression of these selected viruses should be investigated and assessed in order to obtain safe and healthy donor pigs for xenotransplantation studies. Therefore highly sensitive PCR-based methods, real-time PCR and real-time RT-PCR specific for all the viruses listed above, as well as immunological methods measuring virus-specific antibodies by Western blot analysis or ELISA were developed. Recombinant viral proteins were cloned, expressed and chromatographically purified as well as purified virus particles were expanded to be used as antigens. The methods were developed and optimized to screen (i) Göttingen minipigs, a well characterized pig breed which is kept in a specific-pathogen free facility, (ii) Aachen minipigs, a pig breed existing since 2013, (iii) slaughterhouse pigs from a butchery in the north of Berlin and (iv) multiply genetically modified pigs produced especially for xenotransplantation. Human-tropic PERV-A and PERV-B were found in all pigs and pig-tropic PERV-C and recombinant PERV-A/C were found in many pigs. HEV, PCMV, PLHVs and PCV2 were found in a few animals. No transmission of the porcine viruses listed above was observed during the transplantation of genetically modified islet cells into four marmosets. However, when transgenic pig hearts were transplanted into baboons, then PCMV and HEV were found transmitted, despite the fact that the donor pigs were negative when testing blood and antibody response. To avoid future transmissions of porcine viruses, more sensitive detection methods, different time points of testing, and different source materials, including oral and anal swabs, should be used. In the study sensitive and reliable methods for the detection of porcine viruses were developed and those viruses were detected in all tested pig herds. Furthermore, potentially zoonotic viruses like HEV and viruses causing immunosuppression like PCMV, PLHVs and PCV2 are present in pigs for slaughter. Although the expression of these viruses were low, the meat-producing and -processing industry should be aware of the improvement of hygienic standards. The newly developed detection methods are a prerequisite for the selection of virus-free pigs for transplantation trials as well as elimination programs based on treatment, vaccination, Caesarean delivery, early weaning and embryo transfer

Antibody Cross-Reactivity between Porcine Cytomegalovirus (PCMV) and Human Herpesvirus-6 (HHV-6)

Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age

Virus Safety of Xenotransplantation: Prevalence of Porcine Circovirus 2 (PCV2) in Pigs

Porcine circovirus 2 (PCV2) is the causative agent of a whole series of diseases in pigs, called PCV2 diseases (PCVD). The most relevant of them is the systemic disease (PCV2-SD), formerly called post weaning multi systemic wasting syndrome (PMWS). Xenotransplantation using pig cells, tissues and organs is under development to overcome the shortage of human transplants for the treatment of tissue and organ failure. Xenotransplantation requires functional cells, tissues or organs from healthy animals and therefore the donor pigs should be free of PCV2. Selection of PCV2-free animals will also prevent transmission of the virus to the human recipient. Using a PCR method, (i) Göttingen Minipigs, which are well characterised and which were already used in pig to non-human primate xenotransplantations, (ii) newly generated Aachen Minipigs, (iii) genetically modified pigs generated for xenotransplantation, (iv) pigs from a slaughterhouse and (v) pigs from a German farm were screened for PCV2. 50% of the Aachen minipigs and 14% of Göttingen minipigs were PCV2 positive, but the animals were apparently healthy. None of the slaughterhouse animals, the farm animals and the genetically modified animals were positive for PCV2, because they had been vaccinated. The data indicate that PCV2 may be found in healthy pigs even under SPF conditions, and that vaccination is a powerful tool to prevent infection

Microbiological characterization of a newly established pig breed, Aachen Minipigs

Background To alleviate the shortage of human donor organs or tissues for the treatment of organ and tissue failure including diabetes, pigs are considered suitable donor animals. As organs from conventional pigs are usually too large, those from minipigs may be better suited. We recently characterized the Göttingen Minipigs, a breed well characterized concerning the presence of zoonotic microorganisms and found hepatitis E virus (HEV) and porcine cytomegalovirus (PCMV) in some animals. Here, we characterize another minipig, the Aachen Minipig (AaMP), a pig breed recently established close to the town Aachen in Germany. Methods The animals were tested for the prevalence and expression of porcine endogenous retroviruses (PERVs) and the presence of some selected microorganisms, among them HEV, PCMV, and porcine lymphotropic herpesviruses (PLHVs) using highly sensitive and specific PCR and RT-PCR methods. In addition, we screened for antibodies against HEV and PLHV. Results PERV-A, PERV-B, and PERV-C sequences were found in the genome of all Aachen Minipigs. HEV RNA was found by real-time RT-PCR in most, and DNA of PCMV, PLHV-2, and PLHV-3 was found by PCR in some animals. The animals were free of eight other microorganisms tested, but some were seropositive for porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine epidemic diarrhea virus (PEDV). Conclusion Based on medical examinations by veterinarians, the AaMP are in a good health status and seem to harbor only few microorganisms. To improve their status for use as donor pigs in xenotransplantation, the viruses detected might be eliminated by selection of negative animals, Cesarean section, and vaccination

Loyalty and Disavowal in Holy Qur’an Descriptive linguistic study

<span>This research aims to clarify the meaning of loyalty and disavowal from linguistic point of view, between the views of linguists, and the exegetes in the Lexicons and exegeses with analysis and debate. First of all, the researcher Indicates definition of loyalty as stated in the dictionaries, with bringing the examples from the Holy Qur’an and the views of the commentators about this issue, then analysis and discussion. Second, he defines Disavowal in the language, with a point of view from linguists, and interpreters, by analyzing and discussion the examples from the Noble Qur’an. Finally, it illustrates the application of the legal and practical concept of loyalty and disavowal, to enhance the significance of language. The importance of the current study is- according to researcher’s information- due to its doctrinal and sociological matter and nobody precede such a linguistic study of Loyalty. This research approaches the issue, using the method of descriptive and analytical, based on the description and analysis of the phenomenon.</span

Antibody Cross-Reactivity between Porcine Cytomegalovirus (PCMV) and Human Herpesvirus-6 (HHV-6)

Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6