Robert A. Wilkinson

Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

Virally cleansing the pig genome Transplants from pigs could be a solution to a shortage of human organs for transplantation. Unfortunately, porcine endogenous retroviruses (PERVs) are rife in pigs and can be transmitted to humans, risking disease. L. Yang et al. integrated CRISPR-Cas into the pig cell genome, where continuous induction of the Cas9 editing enzyme resulted in the mutation of every single PERV reverse transcriptase gene. This prevented replication of all copies of PERV, viral infection, and transmission to human cells. Science, this issue p. 1101 CRISPR-Cas genome editing is adapted to remove 62 copies of a retrovirus from the porcine genome. The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

111In-Labeled Nonspecific Immunoglobulin Scanning in the Detection of Focal Infection

We performed radionuclide scanning after the intravenous injection of human IgG labeled with indium-111 in 128 patients with suspected focal sites of inflammation. Localization of 111In-labeled IgG correlated with clinical findings in 51 infected patients (21 with abdominal or pelvic infections, 11 with intravascular infections, 7 with pulmonary infections, and 12 with skeletal infections). Infecting organisms included gram-positive bacteria, gram-negative bacteria, Pneumocystis carinii, Mycoplasma pneumoniae, and Candida albicans. No focal localization of 111In-labeled IgG was observed in 63 patients without infection. There were five false negative results, and nine results were unusable. Serial scans were carried out in eight patients: continued localization correctly predicted relapse in six, and the absence of localization indicated resolution in two. To determine whether 111In-labeled IgG localization was specific for inflammation, we studied 16 patients with cancer. Focal localization occurred in 13 of these patients (5 with melanomas, 5 with gynecologic cancers, and 1 each with lymphoma, prostate cancer, and malignant fibrous histiocytoma). No localization was seen in patients with renal or colon cancer or metastatic medullary carcinoma of the thyroid. We conclude that 111In-labeled IgG imaging is effective for the detection of focal infection and that serial scans may be useful in assessing therapeutic efficacy. This technique may also be helpful in the evaluation of certain cancers.

Detection of acute inflammation with 111In-Labeled nonspecific polyclonal IgG

The detection of focal sites of inflammation is an integral part of the clinical evaluation of the febrile patient. When anatomically distinct abscesses are present, lesion detection can be accomplished by standard radiographic techniques, particularly in patients with normal anatomy. At the phlegmon stage, however, and in patients who have undergone surgery, these techniques are considerably less effective. While radionuclide methods, such as Gallium-67 (67Ga)-citrate and Indium-111 (111In)-labeled WBCs have been relatively successful for the detection of early inflammation, neither approach is ideal. In the course of studies addressing the use of specific organism-directed antibodies for imaging experimental infections in animals, we observed that nonspecific polyclonal immunoglobulin G (IgG) localized as well as specific antibodies. Preliminary experiments suggested that the Fc portion of IgG is necessary for effective inflammation localization. Since polyclonal IgG in gram quantities has been safely used for therapy in patients with immune deficiency states, we decided to test whether milligram quantities of radiolabeled IgG could image focal sites of inflammation in humans. Thus far, we have studied a series of 84 patients with suspected lesions in the abdomen, pelvis, vascular grafts, lungs, or bones/joints. In 48 of 52 patients with focal lesions detected by surgery, computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), the IgG scan correctly localized the site, while 31 patients without focal inflammation had no abnormal focal localization of the radiopharmaceutical. Four patients had false negative scans and one patient had a false positive scan. For this small series, the overall sensitivity and specificity were 92% and 95%, respectively. In this report, we review our experience with this exciting new agent.

Reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation

Background. Xenotransplantation using pigs as the source species for organs carries a potential risk for transmission and activation of porcine herpesviruses. Activation of porcine cytomegalovirus (PCMV) in pig-to-baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy (CC). Methods. To further investigate the role of PCMV activation in the occurrence of CC, a strategy to exclude PCMV from the donor was developed. To exclude PCMV, piglets were early-weaned and raised separated from other swine. These piglets were used as donors in an experimental protocol of pig-to-baboon heart xenotransplantation. Results. Early weaning of piglets was successful in excluding PCMV. Use of PCMV-free cardiac porcine xenografts in baboons resulted in prolonged graft survival and prevented consumptive coagulopathy in all recipients. Conclusions. The use of PCMV-free cardiac grafts is beneficial in reducing the direct effects of PCMV activation in the graft (tissue damage) and the indirect effects of PCMV activation in the recipient (consumptive coagulopathy).

Genomic presence of recombinant porcine endogenous retrovirus in transmitting miniature swine

The replication of porcine endogenous retrovirus (PERV) in human cell lines suggests a potential infectious risk in xenotransplantation. PERV isolated from human cells following cocultivation with porcine peripheral blood mononuclear cells is a recombinant of PERV-A and PERV-C. We describe two different recombinant PERV-AC sequences in the cellular DNA of some transmitting miniature swine. This is the first evidence of PERV-AC recombinant virus in porcine genomic DNA that may have resulted from autoinfection following exogenous viral recombination. Infectious risk in xenotransplantation will be defined by the activity of PERV loci in vivo.

Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model.

Background Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our &agr;1,3-galactosyltransferase gene knockout swine herd. Methods Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed. Results Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. Conclusions Decreased survivals of &agr;1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term human-porcine chimeric mice

Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans.

Reduced Efficacy of Ganciclovir Against Porcine and Baboon Cytomegalovirus in Pig-to-Baboon Xenotransplantation

In pig‐to‐baboon xenotransplantation, porcine cytomegalovirus (PCMV) causes viremia, consumptive coagulopathy, and tissue‐invasive disease. Baboon cytomegalovirus (BCMV) is associated with invasive disease in xenograft recipients. The efficacy of prophylaxis with intravenous ganciclovir (GCV) was studied for prevention of PCMV and BCMV infections in pig‐to baboon xenotransplantation. GCV prophylaxis did not alter the incidence of BCMV activation in recipients, but reduced the amount of virus in tissues (mean 8.38 × 102 vs. 3.24 × 105 copies/µg DNA without treatment) and prevented tissue‐invasive infections. PCMV viral loads were unaltered by GCV prophylaxis (8.36 × 108 copies/µg DNA without prophylaxis vs. 1.20 × 109 copies/µg DNA with prophylaxis). In vitro, PCMV was relatively resistant to GCV [90% inhibitory concentration (IC90) of 10 µm, IC50 = 3 µm], acyclovir (100 µm), and leflunomide (not achievable). Only cidofovir (IC90 1 µm) and foscarnet (IC90 100 µm) might have therapeutic efficacy for PCMV in vivo in achievable concentrations, although these agents often carry significant toxicity in transplant recipients. GCV has limited activity against BCMV and no therapeutic efficacy against PCMV at standard doses in vivo. GCV and other antiviral agents have limited activities against PCMV in vitro. Breeding of PCMV‐free xenograft donors may be necessary to prevent PCMV infections in clinical trials.

Utility of the indium 111-labeled human immunoglobulin G scan for the detection of focal vascular graft infection

The ability to diagnose and localize vascular graft infections has been a major challenge. Recent studies in animal models and humans with focal bacterial infection have shown that radiolabeled, polyclonal, human immunoglobulin G accumulates at the site of inflammation and can serve as the basis for an imaging technique. This study investigated this new technique for the diagnosis and localization of vascular graft infections. Twenty-five patients with suspected vascular infections involving grafts (22), atherosclerotic aneurysms (2), and subclavian vein thrombophlebitis (1) were studied. Gamma camera images of the suspected area were obtained between 5 and 48 hours after intravenous administration of 1.5 to 2.0 mCi (56 to 74 mBq) of indium 111-labeled, human, polyclonal immunoglobulin G. Scan results were interpreted without clinical information about the patient and were subsequently correlated with surgical findings, other imaging modalities, and/or clinical follow-up. In 10 of 10 patients found to have positive scan results, localized infections were confirmed at the involved sites. In 14 of 15 patients whose scan results were interpreted as negative, no vascular infections were identified at follow-up. The patient with false-negative results and recurrent bacteremia from an aortoduodenal fistula was found to have a negative scan outcome at a time when his disease was quiescent. These data suggest that nonspecific, human, indium 111-labeled immunoglobulin G scanning can be a useful noninvasive means of localizing vascular infections.

Effect of isoelectric point on biodistribution and inflammation: imaging with indium-111-labelled IgG.

Electrostatic effects play an important role in protein interactions and may alter the biodistribution of antibodies. To study the effect of molecular charge on the biodistribution and infection imaging properties of human polyclonal immunoglobulin G (IgG), its iso electric point was varied by changing the level of diethylene triamine penta-acetic acid (DTPA) substitution: 0.8, 0.9, 3.7, 5.1 and 5.9 DTPA/IgG. Biodistributions of the different IgG preparations were determined at 10 min, 1, 6, 24, and 48 h post injection in normal rats, and infection imaging properties were determined in rats withEscherichia coli thigh infections. The biodistribution was significantly affected by pl. The immunoglobulin preparations with 0.9 and 3.7 DTPA/IgG showed faster clearance from the circulation and generally lower accumulation in most organs. The images had a target-to-background ratio of approximately 1.3–2.3:1. These results suggest that even though targeting is not affected by the level of DTPA substitutions, preparations with 0.9 and 3.7 DTPA/IgG may be superior imaging agents because of reduced accumulation by background organs.