Monitoring pCMV and cytokine levels is crucial for recipient survival in pig-to-cynomolgus Xenotransplantation

Introduction: Given the global shortage of donor organs, pigs have been proposed as a promising source due to their similar organ size to humans. However, it has been reported that the presence of porcine cytomegalovirus (pCMV) significantly reduces the survival rate of immunosuppressed recipients of xenotransplants, and sensitive detection methods are necessary due to the virus’s latency. Because immunosuppressants weaken the immune system, they increase the risk of opportunistic infections. It is important to consider the status of donor’s organ, particularly regarding pCMV infection. We screened for the presence of pCMV using RT-PCR to study the association between increases in proinflammatory cytokines induced by pCMV infection in the recipient’s blood sample and decreased survival rate of the recipients. Methods: Twenty-one cynomolgus macaques received kidney xenotransplantation from genetically modified pigs, and an anti-CD154 Ab immunosuppression protocol was used. Blood samples were collected from the pig donors before transplantation and at regular intervals before, during, and after transplantation from the cynomolgus monkey recipients. DNA was extracted from peripheral blood mononuclear cells (PBMCs) and serum, and real-time PCR was used to detect pCMV using the ViroReal kit. Cytokines were also measured in cynomolgus sera using the LEGENDplex NHP Inflammation Assay Kit. Results: Cynomolgus recipients who received a kidney from a donor with pCMV-positive PBMCs (n=10) had a survival time of 16.8 days, while recipients who received a kidney from a donor with pCMV-negative PBMCs (n=11) had a significantly longer survival time of 49.6 days (p<0.01). Out of 8 recipients who survived less than 2 weeks after receiving a kidney from a pCMV positive donor, 7 were found to be pCMV-positive in their blood samples. The level of pCMV positivity increased over time in both PBMC and serum samples taken from the same recipients, indicating viral reactivation in the transplanted pig organs and subsequently, circulation of virus particles through the bloodstream of the recipients. Therefore, monitoring of pCMV using RT-PCR before and after xenotransplantation is crucial. In addition, the presence of pCMV was associated with increased levels of cytokines, including IL-6, IL-10, IP-10, TNF-a, IFN-g, and MCP-1, which were found to affect the survival of the recipients. Conclusion: In conclusion, accurate monitoring using RT-PCR is important for the detection of pCMV in pig-to-cynomolgus xenotransplantation, and the pCMV positivity is highly correlated with recipient survival. Furthermore, the presence of pCMV is also associated with increased cytokines that affect recipient survival, suggesting the importance of monitoring both pCMV and cytokine levels before and after transplantation. HI20C0056. References: 1. Denner, J., Längin, M., Reichart, B. et al. Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival. Sci Rep. 10, 17531 (2020). 2. Denner J. Xenotransplantation and porcine cytomegalovirus. Xenotransplantation. 2015: 22: 329–335.