Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

Application of Methods Detecting Xenotransplantation-Relevant Viruses for Screening German Slaughterhouse Pigs.

Detection methods have been developed to prevent transmission of zoonotic or xenozoonotic porcine viruses after transplantation of pig organs or cells to the recipient (xenotransplantation). Eleven xenotransplantation-relevant viruses, including porcine cytomegalovirus, porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses -1, -2, -3 (PLHV-1, 2, 3), porcine parvovirus (PPV), porcine circovirus 2, 3, 4 (PCV2, 3, 4), hepatitis E virus genotype 3 (HEV3), porcine endogenous retrovirus-C (PERV-C), and recombinant PERV-A/C have been selected. In the past, several pig breeds, minipigs, and genetically modified pigs generated for xenotransplantation had been analyzed using these methods. Here, spleen, liver, and blood samples from 10 German slaughterhouse pigs were screened using both PCR-based and immunological assays. Five viruses: PCMV/PRV, PLHV-1, PLHV-3, and PERV-C, were found in all animals, and PCV3 in one animal. Some animals were latently infected with PCMV/PRV, as only virus-specific antibodies were detected. Others were also PCR positive in the spleen and/or liver, indicative of an ongoing infection. These results provide important information on the viruses that infect German slaughterhouse pigs, and together with the results of previous studies, they reveal that the methods and test strategies efficiently work under field conditions.

An Approach to Controlling Inflammation and Coagulation in Pig-to-Baboon Cardiac Xenotransplantation.

INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

The Endothelial Glycocalyx in Pig-to-Baboon Cardiac Xenotransplantation-First Insights.

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface-the endothelial glycocalyx-are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.

Limited availability of methods for the detection of xenotransplantation-relevant viruses in veterinary laboratories.

BACKGROUND: The German Xenotransplantation Consortium is in the process to prepare a clinical trial application (CTA) on xenotransplantation of genetically modified pig hearts. In the CTA documents to the central and national regulatory authorities, that is, the European Medicines Agency (EMA) and the Paul Ehrlich Institute (PEI), respectively, it is required to list the potential zoonotic or xenozoonotic porcine microorganisms including porcine viruses as well as to describe methods of detection in order to prevent their transmission. The donor animals should be tested using highly sensitive detection systems. I would like to define a detection system as the complex including the actual detection methods, either PCR-based, cell-based, or immunological methods and their sensitivity, as well as sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls. Lessons learned from the identification of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) in the xenotransplanted heart in the recipient in the Baltimore study underline how important such systems are. The question is whether veterinary laboratories can supply such assays. METHODS: A total of 35 veterinary laboratories in Germany were surveyed for their ability to test for selected xenotransplantation-relevant viruses, including PCMV/PRV, hepatitis E virus, and porcine endogenous retrovirus-C (PERV-C). As comparison, data from Swiss laboratories and a laboratory in the USA were analyzed. Furthermore, we assessed which viruses were screened for in clinical and preclinical trials performed until now and during screening of pig populations. RESULTS: Of the nine laboratories that provided viral diagnostics, none of these included all potential viruses of concern, indeed, the most important assays confirmed in recent human trials, antibody detection of PCMV/PRV and screening for PERV-C were not available at all. The situation was similar in Swiss and US laboratories. Different viruses have been tested for in first clinical and preclinical trials performed in various countries. CONCLUSION: Based on these results it is necessary to establish special virological laboratories able to test for all xenotransplantation-relevant viruses using validated assays, optimally in the xenotransplantation centers.

[Xenotransplantation of solid organs]. / Xenotransplantation von Organen.

Transplantation of genetically modified porcine hearts and kidneys could become a solution to the persistent shortage of human organ donors. Progress has been made in genetic engineering of donor pigs, preservation techniques after organ harvesting and immunosuppression using co-stimulation blockade with anti-CD40/CD40L monoclonal antibodies. Progress has also been made in in the development of methods that detect pathogenic porcine viruses and prevent their transmission to the recipient. As normal land breed pig organs continue to grow in the recipient to their original size, different pig breeds (such as Auckland Island pigs) are now used which reach a final size suitable for humans. Alternatively, a knock-out of the growth hormone receptor gene has been established, e.g., in the 10GM genetically modified pigs from Revivicor/United Therapeutics, USA. The first clinical pilot studies including patients suffering from terminal heart failure are expected to start in Germany in about 2 years.

Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

Emergence of novel circoviruses in humans and pigs and their possible importance for xenotransplantation and blood transfusions.

BACKGROUND: As sequencing is becoming more broadly available, virus discovery continues. Small DNA viruses contribute to up to 60% of the overall virus load in pigs. Porcine circoviruses (PCVs) are small DNA viruses with a single-stranded circular genome. They are common in pig breeds and have not been properly addressed for their potential risk in xenotransplantation. Whereas PCV1 is non-pathogenic in pigs, PCV2 has been associated with various disease manifestations. Recently two new circoviruses have been described, PCV3 and PCV4. While PCV4 is currently present mainly in Asia, PCV3 is widely distributed, and has been identified in commercial pigs, wild boars, and pigs generated for xenotransplantation. In one case PCV3 was transmitted by pigs to baboons via heart transplantation. PCV3 pathogenicity in pigs was controversial initially, however, the virus was found to be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic inflammation. Inoculation studies with PCV3 infectious clones confirmed that PCV3 is pathogenic. Most importantly, recently discovered human circoviruses (CV) are closely related to PCV3. METHODS: Literature was evaluated and summarized. A dendrogram of existing circoviruses in pigs, humans, and other animal species was created and assessed at the species level. RESULTS: We found that human circoviruses can be divided into three species, human CV1, CV2, and CV3. Human CV2 and CV3 are closest to PCV3. CONCLUSIONS: Circoviruses are ubiquitous. This communication should create awareness of PCV3 and the newly discovered human circoviruses, which may be a problem for blood transfusions and xenotransplantation in immune suppressed individuals.

Screening for Viruses in Indigenous Greek Black Pigs.

The successful advancement of xenotransplantation has led to the development of highly sensitive detection systems for the screening of potentially zoonotic viruses in donor pigs and preventing their transmission to the recipient. To validate these methods, genetically modified pigs generated for xenotransplantation, numerous minipigs and other pig breeds have been tested, thereby increasing our knowledge concerning the pig virome and the distribution of pig viruses. Of particular importance are the porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV) and the hepatitis E virus genotype 3 (HEV3). PCMV/PRV has been shown to reduce the survival time of pig transplants in non-human primates and was also transmitted in the first pig heart transplantation to a human patient. The main aim of this study was to determine the sensitivities of our methods to detect PCMV/PRV, HEV3, porcine lymphotropic herpesvirus-1 (PLHV-1), PLHV-2, PLHV-3, porcine circovirus 2 (PCV2), PCV3, PCV4 and porcine parvovirus 1 (PPV1) and to apply the methods to screen indigenous Greek black pigs. The high number of viruses found in these animals allowed for the evaluation of numerous detection methods. Since porcine endogenous retroviruses (PERVs) type A and B are integrated in the genome of all pigs, but PERV-C is not, the animals were screened for PERV-C and PERV-A/C. Our detection methods were sensitive and detected PCMV/PRV, PLHV-1, PLHV-1, PLHV-3, PVC3 and PERV-C in most animals. PPV1, HEV3, PCV4 and PERV-A/C were not detected. These data are of great interest since the animals are healthy and resistant to diseases.

Porcine endogenous retroviruses in xenotransplantation.

Xenotransplantation using pig cells, tissues or organs is under development to alleviate the shortage of human donor organs. Meanwhile, remarkably long survival times of pig organs in non-human primates have been reported, as well as the functionality of pig kidneys and hearts in brain-dead humans. Most importantly, two transplantations of pig hearts in patients were performed with survival times of the patients of 8 and 6 weeks. Xenotransplantation may be associated with the transmission of porcine microorganisms including viruses to the recipient. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated like other viruses can. PERVs are able to infect certain human cells and therefore pose a risk for xenotransplantation. It is well known that retroviruses are able to induce tumors and immunodeficiencies. However, until now, PERVs were not transmitted in all infection experiments using small animals and non-human primates, in all preclinical xenotransplantation trials in non-human primates and in all clinical trials in humans. In addition, several strategies including antiretrovirals, PERV-specific small interfering RNA, vaccines and genome editing using CRISPR/Cas have been developed to prevent PERV transmission.

Determination of the Copy Number of Porcine Endogenous Retroviruses (PERV) in Auckland Island Pigs Repeatedly Used for Clinical Xenotransplantation and Elimination of PERV-C.

Auckland Island pigs represent an inbred population of feral pigs isolated on the sub-Antarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand; they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human diabetes patients. The animals do not carry any of the xenotransplantation-relevant viruses, and in the first clinical trials, no porcine viruses, including porcine endogenous retroviruses (PERVs) were transmitted to the human recipients. PERVs pose a special risk in xenotransplantation, since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR and primers binding to a conserved region of the polymerase gene (PERVpol), a copy number typical for Western pigs was found. This confirms previous phylogenetic analyses of microsatellites as well as mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as donors in somatic cell nuclear transfer (SCNT), animals were born showing PERVpol copy numbers between 35 and 56. These data indicate that Auckland Island pigs have a similar copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most importantly, PERV-C-free animals were selected and the absence of an additional eight porcine viruses was demonstrated.

Current Status of Cardiac Xenotransplantation: Report of a Workshop of the German Heart Transplant Centers, Martinsried, March 3, 2023.

This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed. Obviously microbiological safety for the recipient and close contacts is essential, and ethical considerations to gain public acceptance for clinical applications are addressed. The first clinical trial will be regulated and supervised by the Paul-Ehrlich-Institute as the National Competent Authority for Germany, and the German Heart Transplant Centers agreed to cooperatively select the first patients for cardiac xenotransplantation.

Comparison of methods for the detection of porcine cytomegalovirus/roseolovirus in relation to biosafety monitoring of xenotransplantation products.

BACKGROUND: The porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV), is widely distributed in pig populations. It has been shown that PCMV/PRV was transmitted by pig xenotransplants to non-human primates, and significantly reduced the survival time of the recipient. PCMV/PRV was also transmitted during the first transplantation of a pig heart into a human patient. PCMV/PRV establishes a lifelong persistent infection (latency) in the host, is difficult to detect in this stage, and consequential poses a threat to future clinical xenotransplantations. Therefore, sensitive and specific methods and goal-oriented strategies how, when, and where to test should be used for screening donor pigs. METHODS: In this study we compared experimentally the PCMV/PRV detection methods including PCR-based (real-time PCR, nested PCR) and immunological methods (Western blot assay, ELISA) recently published by Halecker et al. (Sci. Rep. 2022;12(1):21545) and Fischer et al. (Xenotransplantation 2023:e12803). We also compared the antigens used for antibody detection (a recombinant protein and synthetic peptides corresponding to a conserved region of the glycoprotein B, gB). RESULTS: The published methods can be used for screening donor pigs, with the results being similar. The antigens used for the detection of PCMV/PRV-specific antibodies are almost identical and give comparable results. Overall, the optimal diagnostic tests, the samples used for testing and the time of sampling play a crucial role in preventing the transmission of PCMV/PRV during xenotransplantation. CONCLUSION: Sensitive methods are available to screen donor pigs for PCMV/PRV, but a rational application of a combination of PCR-based and immunological methods as well as rational detection strategies are important for the detection of the virus during latency.

The large extracellular loop is important for recruiting CD63 to exosomes.

Exosomes are small extracellular vesicles that are secreted from cells. To characterize exosome fraction marker proteins of the tetraspanin family in particular, CD9, CD63, and CD81 are routinely used. CD63 expression constructs were employed to investigate the influence of the large extracellular loop (LEL) of CD63 on sorting into exosomes. When the LEL of CD63 fused with mCherry was deleted, the protein was no longer found in the purified exosome fraction. This finding demonstrates the importance of the LEL sequence for the recruitment of CD63 into exosomes.

Sensitive detection of lipopolysaccharides by monitoring of interleukin-10 secretion from human PBMCs.

Lipopolysaccharide (LPS) contaminations may falsify immunological experiments and are crucial for pharmaceutical products because they cause life-threatening immune reactions. Here, we present interleukin-10 (IL-10) as a reliable marker to measure LPS contents when the readout of pro-inflammatory cytokines is not favored. This animal free source assay is able to detect LPS with a limit of detection (LOD) of 0.024 EU/ml by monitoring IL-10 secretions from isolated human peripheral blood mononuclear cells (PBMCs).

Evidence for Microchimerism in Baboon Recipients of Pig Hearts.

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials.

First virological and pathological study of Göttingen Minipigs with Dippity Pig Syndrome (DPS).

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.