Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.     

No evidence for infection of human cells with porcine endogenous retrovirus (PERV) after exposure to porcine fetal neuronal cells

BACKGROUND: Recent demonstration of human cell infection in vitro with porcine endogenous retrovirus (PERV) has raised safety concerns for new therapies that involve transplantation of pig cells or organs to humans. To assess better the specific risk that may be associated with the transplantation of fetal pig neuronal cells to the central nervous system of patients suffering from intractable neurologic disorders (Parkinson's disease, Huntington's disease, and epilepsy), we have performed studies to determine whether there is evidence for in vivo or in vitro transmission of PERV from fetal pig neuronal cells to human cells. METHODS: Ventral mesencephalon (VM) and lateral ganglionic eminence cells were isolated from fetal pigs and transplanted into patients with neurological conditions as part of clinical studies. Blood samples taken from patients at various time points posttransplant were tested for evidence of PERV. In vitro studies to test for PERV infection of human cells after cocultivation with either fetal porcine ventral mesencephalon or porcine fetal lateral ganglionic eminence cells were also performed. RESULTS: We found no evidence of PERV provirus integration in the DNA from PBMC of 24 neuronal transplant recipients. In addition, no PERV was released from cultured fetal porcine neuronal cultures, and there was no transfer of PERV from fetal pig neuronal cells to human cells in vitro. CONCLUSIONS: Our results demonstrate by both examination of transplant patient blood samples and in vitro studies that there is no evidence for transmission of PERV from porcine fetal neural cells to human cells.     

AN69 hollow fiber membrane will reduce but not abolish the risk of transmission of porcine endogenous retroviruses

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supematant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.     

Evidence of Absence of Porcine Endogenous Retrovirus (PERV) Infection in Patients Treated with a Bioartificial Liver Support System

Porcine endogenous retrovirus (PERV) genomes are present in all pig cells. In this retrospective study, we assessed PERV infectivity in 28 patients treated with an extracorporeal bioartificial liver (HepatAssist system) that includes a membrane device containing porcine hepatocytes. All patients tested negative for PERV using polymerase chain reaction analysis of peripheral blood mononuclear cells (PBMC) collected up to 5 years after treatment. In vitro results showed that the membrane decreased the risk of PERV transmission by a factor of 105, and porcine hepatocytes did not produce infectious PERV in co-cultures with human cell line 293. Our results do not support the presence of PERV infection in patients treated with this porcine hepatocyte-based bioartificial liver.     

Porcine endogenous retrovirus encodes xenoantigens involved in porcine cellular xenograft rejection by mice

BACKGROUND: Identification of the antigens that stimulate transplant rejection can help develop graft-specific antirejection strategies. The xenoantigens recognized during rejection of porcine cellular xenografts have not been clearly defined, but it has been assumed that major histocompatibility complex (MHC) xenoantigens are involved. METHODS: The role of porcine endogenous retrovirus (PERV) as a source of xenoantigens was examined. The authors used morphometry to compare the kinetics of swine leukocyte antigen (SLA) pig thyroid xenograft rejection in control mice and mice immunized with PERV PK15 cells (porcine kidney epithelial cells), PERV SLA pig peripheral blood lymphocytes (PBL), PERV virions purified from PK15 cells, and PERV or PERV A pseudotypes produced from infected human 293 cells. The tempo of rejection for cellular xenografts of PERV A pseudotype-producing human 293 cells, uninfected human 293 cells, and PK15 cells in PERV-preimmunized and control mice was also compared. RESULTS: Mice immunized with PK15 cells rejected pig thyroid xenografts significantly faster at day 5 than control mice and mice immunized with pig PBL. This correlated with the amount of PERV RNA and virions produced, but not with the amount of SLA class I MHC expressed by PK15 cells. Immunization of mice with PERV virions purified from porcine PK15 cells and with PERV virions or PERV A pseudotypes produced by human 293 cells also induced accelerated xenograft rejection by 5 days. Accelerated rejection induced by virus pretreatment was CD4 T-cell dependent and restricted to PERV-expressing cellular xenografts of porcine or nonporcine origin. CONCLUSIONS: PERV acts as a significant source of xenoantigens that target porcine cellular xenografts for rejection.     

利用人源化小鼠研究组织工程材料的体内免疫原性

目的建立并利用人源化小鼠模型,研究组织工程材料的体内免疫原性。方法利用NOD／SCID小鼠（非肥胖型糖尿病鼠与重症联合免疫缺陷鼠反交鼠模型）．通过腹腔注射人外周血单个核细胞（PBMC）（100×10^6）建立人源化小鼠,在人源化小鼠皮下植入异体人皮肤（阳性对照）、人体脂肪干细胞与脱钙骨构建的组织工程骨（实验组）、单纯脱钙骨材料（阴性对照）。在植入后1周、2周、3周、4周,取小鼠尾静脉血,流式细胞仪检测人CD4^＋和CD8^＋淋巴细胞比例。4周后,取小鼠的移植局部皮肤进行HE染色分析,同时检测脾细胞人CD4^＋和CD8^＋淋巴细胞比例。结果NOD／SCID小鼠腹腔注射人PBMC4周内,在外周血和脾中均可测到人CD4^＋和CD8^＋淋巴细胞。病理分析结果显示：在人源化小鼠皮下植入的组织工程材料及成体干细胞构建的组织工程骨组未见炎性细胞浸润;而单独植入人皮肤组可见明显的炎性细胞浸润。结论人源化小鼠可作为组织工程材料体内免疫原性的研究的良好模型。     

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Di Nicuolo G, D’Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long‐term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. © 2010 John Wiley & Sons A/S. Abstract: Background: Clinical use of porcine cell–based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow‐up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Methods: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center‐BAL (AMC‐BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real‐time polymerase chain reaction assay has been used. Results: Eight patients who received a liver transplant after AMC‐BAL treatment are still alive under long‐term pharmacological immunosuppression. The current clinical follow‐up ranges from 5.6 to 8.7 yr after BAL treatment. A new q‐real‐time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥5 copies per 1 × 10⁵ PBMCs. The linear dynamic range was from 5 × 10⁰ to 5 × 10⁶ copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Conclusion: Up to 8.7 yr after exposure to treatment with porcine liver cell–based BAL, no PERV infection has been found in long‐term immunosuppressed patients and in HCWs by a new highly sensitive and specific q‐real‐time PCR assay.     

APOBEC3 proteins and porcine endogenous retroviruses

Xenotransplantation of porcine cells, tissues, and organs offers a solution to overcome the shortage of human donor materials. In addition to the immunological and physiological barriers, the existence of numerous porcine microorganisms including viruses poses a risk for xenozoonosis. Three classes of functional gamma-type porcine endogenous retroviruses (PERV) have been identified, whereby functional polytropic PERV-A and PERV-B infect human embryonic kidney (HEK 293) and other cell lines in vitro. In the course of risk assessment for xenotransplantation the capacity of human cells to counteract PERV infections should be analyzed. Primates and other mammals display different means of protection against viral infections. APOBEC3 proteins which are cytidine deaminases and a part of the intrinsic immunity mediate potent activity against a wide range of retroviruses including murine leukemia viruses (MLV). As PERV and MLV belong to the same genus, we raised the question as to whether PERV is affected by APOBEC3 proteins. Initial data indicate that human and porcine cytidine deaminases inhibit PERV replication, thereby possibly reducing the risk for infection of human cells by PERV as a consequence of pig-to-human xenotransplantation.     

Absence of PERV specific humoral immune response in baboons after transplantation of porcine cells or organs

Xenotransplantation of pig organs seems a promising way of overcoming the prevailing limitation on allotransplantation due to donor numbers. However, as porcine endogenous retroviruses (PERVs) can infect human cells in vitro, there is substantial concern regarding the risk of a PERV infection in xenogeneic transplant recipients. Cultured porcine endothelial cells, stimulated peripheral blood mononuclear cells, and pancreatic islet cells can release PERV infectious for human cells in vitro, but it is currently unknown whether PERV is released in vivo, whether these viral particles can infect the transplant recipient, and whether they are pathogenic. In a retrospective study 15 immunosuppressed baboons were tested for a specific immune response against PERV after transplantation of porcine endothelial cells, mononuclear blood cells, and lungs. Anti-PERV antibody expression was analyzed with peptide-based, enzyme-linked immunosorbent assays and highly sensitive Western Blot assays. This xenotransplantation study using nonhuman primates found no evidence of PERV specific humoral immune response. Our data suggest that no productive PERV infection and no continuous PERV release takes place in the nonhuman primates analyzed in this study.     

Long-term survival of HLA-DR15+ pig skin in SCID mice after reconstitution with human peripheral blood mononuclear cells and under short-term immunosuppression

In allotransplantation, donor-recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor-recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15(-) hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15(-) human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.