Mitigating Cross-Species Viral Infections in Xenotransplantation: Progress,Strategies,and Clinical Outlook

Abstract

Xenotransplantation holds great promise as a solution to address the critical shortage of organs, but it raises concerns regarding the potential transmission of porcine viruses to recipients, leading to infections and even zoonotic diseases. Data used in this review were mainly from literature of Pubmed database. Keywords included xenotransplantation, infection, virus, and epidemiology. The original articles and critical reviews selected were relevant to this review’s theme. We review the major viral infections of concern in xenotransplantation, their risk of transmission, diagnosis, treatment, and ways to prevent infection. Then, we pivot to a comprehensive overview of the current status of xenotransplantation. In addition, we offer our own insights and recommendations for propelling xenotransplantation forward, transitioning from preclinical experiments to the critical phase of clinical trials. Viral infections pose considerable safety concerns within xenotransplantation, particularly with the possibility of emerging or currently unidentified viruses. Clinical trials serve as a crucial platform to progress the safety standards of xenotransplantation. However, further studies and dedicated efforts are required to effectively translate findings into practical applications that can improve safety measures in this field.

Introduction

A persistent worldwide shortage of organs from deceased human donors for transplantation plagues patients with end-stage organ failure ¹ . For instance, a survey by the International Society for Heart and Lung Transplantation (ISHLT) highlights the stark reality that a vast number of end-stage heart failure patients eagerly await a heart transplant, with fewer than 4% fortunate enough to receive one ² . Xenotransplantation, the transfer of living cells, tissues, or organs from genetically engineered pigs, emerges as a promising alternative to alleviate this organ shortage crisis^3–5. In 2022, a historic milestone was achieved at the University of Maryland Medical Center, where the first-ever gene-edited pig heart was successfully transplanted into a human recipient, yielding encouraging data ⁶ . This remarkable achievement has significantly bolstered the confidence of scientists in the field. Nevertheless, the successful application of xenografts carries an inherent risk—viral infection ⁷ . Infections can have dire consequences, impacting survival rates, exacerbating inflammatory responses, and triggering immune rejection in allotransplantation^8–10. Anticipating the prevalence of infections in xenotransplantation, it is crucial to recognize that these infections and their pathogenic effects may be even more pronounced, given the imperative need for immunosuppression in xenotransplantation ¹¹ .

Viruses

Drawing from experiences in allotransplantation and insights from preclinical models, viral infections loom as our foremost concern, with the potential to even trigger zoonotic transmissions. This concern finds stark confirmation in the case of the recipient of the world’s first pig heart xenotransplantation, whose unfortunate demise was suspected to result from a porcine cytomegalovirus (PCMV) infection ¹² . This sobering event underscores the inherent perils of viral infections in xenotransplantation. Following the guidelines outlined by the American Society of Transplantation, we focus our analysis on three key viruses: PCMV, porcine lymphotropic herpesvirus (PLHV), and porcine endogenous retrovirus (PERV) ¹³ . Our investigation spans a comprehensive exploration of these viruses, encompassing their epidemiology, modes of transmission, monitoring techniques, and more.

Porcine Cytomegalovirus

PCMV, officially known as suid beta herpesvirus 2 (SuBHV2) in accordance with the classification by the International Committee on Taxonomy of Viruses (ICTV), belongs to the Roseolovirus genus, signifying its place within the Beta herpesvirinae subfamily^14,15. Analysis of its genome sequence has revealed a total length of 128,367 base pairs, encompassing 79 predicted open reading frames (ORFs) ¹⁴ . PCMV exhibits a widespread presence among pig populations worldwide. For instance, in the Hunan and Sichuan provinces of China, the prevalence of PCMV-positive pigs stands at 96.4% and 84.4%, respectively^16,17. PCMV has been linked to various ailments in pigs, including pneumonia, rhinitis, small body size, miscarriages in female pigs, and edema of the heart and other organs ¹⁸ . Notably, PCMV has been associated with consumptive coagulopathy in a recent study ¹⁹ . In the context of xenotransplantation, PCMV infections have proven detrimental, leading to a reduction in graft survival time. Previous investigations have demonstrated that the presence of PCMV during porcine heart transplantation in baboons resulted in a significant decrease in survival times, with durations dwindling from 33 days to 20 days and from 195 days to 30 days, respectively^20,21. Moreover, in 2022, a groundbreaking xenotransplantation at the University of Maryland in Baltimore involved a pig heart transplant into a human patient. This case raised concerns that PCMV may have contributed to the patient’s unfortunate demise, given that the virus potentially entered the recipient’s body along with the transplanted organ ¹² . Similar to other herpesviruses known for their resilience within host organisms, PCMV can undergo reactivation in vivo under conditions of stress ²² . The study had reported the presence of antibody cross-reactivity between PCMV and human herpesvirus 6 (HHV-6) ²³ . While PCMV is unlikely to infect human cells or replicate within the human body, uncertainties persist regarding its potential to induce diseases akin to HHV-6, like roseola infantum, or reduce the survival time of the pig transplant ²⁴ . These findings underscore the critical significance of PCMV in the context of xenograft survival.

Numerous polymerase chain reaction (PCR) methods are at our disposal for the detection of PCMV, some of which yield longer amplicons, enhancing our ability to sequence and classify the detected PCMVs effectively ²⁵ . However, it is important to note that certain PCR methods may occasionally yield false negatives ²⁶ . Therefore, there is a need for the adoption of more sensitive PCR techniques. New diagnostic approaches, such as nested PCR and duplex real-time PCR systems, have been developed with improved parameters, significantly bolstering their sensitivity and accuracy^25,27. In addition to PCR-based methods, immunological techniques for PCMV detection have also seen advancements. Using recombinant proteins corresponding to the two domains of PCMV glycoprotein gB as antigens, researchers have employed Western blot technology to analyze the presence of PCMV-specific antibodies ²⁸ . Furthermore, an indirect-blocking enzyme-linked immunosorbent assay (ELISA) method designed to detect the gB epitope has demonstrated impressive specificity at 98% and remarkable sensitivity at 97.8% ²⁹ . In terms of the detection timeframe, real-time PCR offers a distinct advantage, as it facilitates the early detection of PCMV presence in piglets compared to antibody testing^12,26.

Porcine Lymphotropic Herpesvirus

PLHV-1, PLHV-2, and PLHV-3 belong to the gamma herpesvirus family and are prevalent among pigs. A survey conducted in a densely populated pig area in Northern Italy revealed the widespread presence of PLHVs, with prevalence rates of 28.97%, 10.79%, and 4.54% for PLHV-1, PLHV-2, and PLHV-3, respectively. These viruses were not only detected in various pig tissues but were also associated with specific clinical conditions ³⁰ . PLHV confirmed to infect persistently porcine B cell line L23^31,32. While PLHV is generally benign in its natural host, it can pose a significant health threat when transmitted to other species, causing severe diseases ³³ . Surprisingly, no direct link between PLHV and swine diseases has been established thus far. However, research has indicated that PLHV-1 can induce posttransplant lymphoproliferative disease (PTLD) in minipigs^34,35. The primary mode of PLHV transmission is horizontal, but there is also a risk of vertical transmission from parent to offspring^33,36. Currently, no treatments or vaccines are available for these three PLHVs.

Even strategies like early weaning and colostrum feeding have proven ineffective in eliminating PLHV ³⁷ . Although practices such as cesarean section and barrier maintenance have reduced PLHV infection rates from 80% to as low as 3% to 12.8%, they are not foolproof ³⁸ , necessitating the development of sensitive detection methods and novel approaches for PLHV elimination.

PLHV presence has been systematically examined and quantified using PCR assays, with a particular focus on specific real-time PCR techniques^30,39. These assays have strategically employed primers and probes targeting key regions within the DNA polymerase gene and the gene responsible for encoding glycoprotein B ³³ . In addition to PCR-based approaches, the detection of antibody responses to recombinant glycoprotein B of PLHV-1 has been explored using both Western blot assays and ELISA methods^40,41. These comprehensive techniques have significantly contributed to our understanding of PLHV dynamics and its impact on host organisms.

Porcine Endogenous Retroviruses

Numerous viruses have been identified in both pigs and humans, with PERVs standing out as particularly noteworthy. PERVs are known to be transmissible from pig cells to human cells, making them a subject of significant concern ⁴² . There are three types of PERVs: PERV-A and PERV-B, which are integrated into the genome of all pigs, and PERV-C, which is present in most, but not all, pigs. While PERV-A and PERV-B are polytropic and capable of infecting various human cell types, PERV-C is ecotropic and exclusively infects porcine cells. Intriguingly, PERV-A and PERV-C can combine to form PERV-A/C recombinants. These recombinants exhibit a similar ability to infect human cells as PERV-A but have been shown to have a higher replication rate than PERV-A alone ⁴³ . PCR amplification technology has proven effective in detecting PERVs in the genomes of four major miniature pig breeds in China ⁴⁴ . Over time, PERVs have been observed to remain active within their host organisms, with the number of copies increasing ⁴⁵ . Notably, PERVs carry the potential to induce various health issues, including the development of tumors, leukemias, and neurodegenerative diseases^46,47. Recent research has also revealed that PERVs can stimulate the production of the pro-inflammatory chemokine CXCL10 in human monocytes and monocyte-derived primary cells, thereby enhancing the innate immune response within the host ⁴⁸ . To date, no transmission of PERVs has been observed in clinical trials or preclinical trials^49,50. There are several possible explanations for this absence of transmission. It is conceivable that PERVs are not released from the graft, or they may be suppressed by intracellular restriction factors and the innate immune response within the recipient ⁵¹ .

The patients selected for the clinical porcine islet xenotransplantation were nonimmunocompromised individuals with diabetes. This characteristic might have contributed to preventing the spread of PERV ⁵⁰ . It has been demonstrated that PERVs can infect human cells^52,53 and integrate into human genome in cell culture ⁵⁴ . This raises concerns regarding the possibility of PERVs being transmitted through a transplant patient and potentially becoming infectious to individuals in contact with that patient, posing a potential source for a widespread epidemic^13,55. In addition, microchimerism, which can be challenging to distinguish from PERV infection, has been reported in the initial human xenograft trials and in most preclinical trials involving nonhuman primates^56,57. These findings underscore the critical importance of continuous monitoring and accurate identification of PERVs in xenotransplantation research.

Given the significant variation in PERV copy numbers across different pig breeds, employing PCR to quantify PERV copy numbers in various pig breeds becomes invaluable for selecting the most suitable candidates for xenotransplantation^44,58. Among the available techniques, droplet digital PCR (ddPCR) stands out as the most commonly used method ⁴⁴ . Furthermore, the presence of PERV particles can be visually confirmed through electron microscopy, providing an additional means of detection ⁵⁹ . An innovative approach proposed in a study is the immunoperoxidase assay (IPA), designed to detect viral proteins in infected cells and antibodies against PERV in the serum of the infected host ⁶⁰ . These diverse methodologies offer essential tools for assessing and managing the risk associated with PERVs in xenotransplantation settings.

Elimination of Porcine Viruses

Control of Pig Donors

Selection of pig herds

Notably, not all pigs harbor specific pathogens, underscoring the critical need to identify and assess the pathogens present in pigs for the purpose of selecting suitable donors in xenotransplantation endeavors. Establishing an “exclusion list” serves as a fundamental foundation for the screening of potential pig donors, and it necessitates periodic revision in response to evolving global swine infection epidemiology and evolving clinical experience ⁷ . As an illustrative example, the selection of pigs with diminished expression of PERV-A and PERV-B or those lacking PERV-C can be a proactive measure to prevent the binding of PERV-A and PERV-C ⁶¹ . Such negative pigs represent viable candidates for direct use in xenotransplantation procedures, thus mitigating the risk associated with PERV transmission.

Medicines

In situations where negative pigs are not readily available, an alternative approach involves the selection of pigs with low viral loads, followed by treatment using vaccination or antiviral drugs ⁶² . Specifically, PCMV can be effectively inhibited by various antiviral drugs, including ganciclovir (a synthetic analogue of 20-deoxy-guanosine), cidofovir (employed in the treatment of HCMV-induced retinitis in humans), foscarnet (functioning as a structural mimic of the anion pyrophosphate, selectively inhibiting the pyrophosphate binding site on viral DNA polymerase), acyclovir (a guanosine analogue), and valaciclovir (a prodrug of acyclovir) ⁶³ . Studies have demonstrated that both ganciclovir and cidofovir exhibit superior efficacy in inhibiting PCMV replication when compared to foscarnet and acyclovir ⁶⁴ . It is worth noting that as of now, there have been no reports of successful anti-PCMV vaccines. Conversely, for PERV, inhibitors targeting viral reverse transcriptase and integrase have shown effectiveness. Among these, azidothymidine (AZT) stands out as the most potent inhibitor of reverse transcriptase, while integrase inhibitors have demonstrated significant efficacy against PERV^65–67. Effective neutralizing antibodies can be induced when different animal species are immunized with PERV’s recombinant transmembrane envelope protein p15E and envelope glycoprotein gB70 ⁶⁸ . These antibodies recognize an epitope in the p15E fusion peptide proximal region (FPPR), termed E1, and an epitope in the membrane proximal external region (MPER), termed E2^69,70. Nonetheless, it is imperative to conduct extensive analyses using diverse animal models to comprehensively evaluate the impact and effectiveness of PERV vaccines.

Cesarean delivery, colostrum deprivation, and early weaning

When negative animals are not available and vaccines or antiviral drugs prove ineffective against viruses with low viral loads, there are alternative methods for virus elimination. Some viruses are transmitted vertically, highlighting the importance of preventing the vertical transmission of viruses from sows to piglets. Studies have confirmed that practices such as early weaning, colostrum deprivation, and cesarean delivery can effectively eliminate the PCMV virus in pigs^37,71. However, it is important to note that these methods do not result in complete clearance of the PLHV virus, as experimental results have indicated the continued presence of PLHV viral DNA in early weaned pigs^36,37.

Biosecurity of pig farms

Once the virus is successfully eliminated, it is imperative to maintain the animal in strict isolation to prevent de novo infections or re-entry of the virus. For instance, certain viruses like Hepatitis E virus (HEV) can persist in drinking water, feces, and buildings^72,73. Therefore, donor pigs should be raised and kept in biosecure facilities that isolate them from the external environment. These facilities should provide filtered air, sterilized water, and irradiated food certified to be free of any mammalian protein. All materials entering these facilities must undergo autoclaving, and personnel should enter through showers and wear specialized clothing ⁷⁴ . Only through rigorous supervision and control of all aspects can donor pigs attain the status of designated pathogen-free (DPF).

RNA Interference

RNA interference (RNAi) represents a swift and efficient method for gene expression suppression. This process involves two primary steps: initially, double-stranded RNA (dsRNA) is cleaved into small interfering RNAs (siRNAs) through the activity of bacterial ribonuclease III (RNase III)-like enzymes. Subsequently, these siRNAs associate with the RNA-induced silencing complex (RISC) and facilitate the degradation of target messenger ribonucleic acid (mRNA)^26,75. In the context of PERV, its expression can be significantly reduced through the use of siRNA molecules that correspond to various segments of viral genes such as gag, pol, and env. The most potent sequences are carefully selected and expressed as short hairpin RNA (shRNA) using the polymerase III vector system. This approach enables the continuous inhibition of PERV replication^76–78. PERV-specific shRNA has demonstrated the ability to reduce PERV expression both in vitro (within PERV-producing human cells) and in vivo (in transgenic pigs engineered to express PERV-specific shRNA) ⁷⁹ . Such interventions contribute to enhancing the safety of xenotransplantation procedures. In addition, some research findings suggest that certain immunosuppressant agents can reduce PERV expression in vitro without synergistic or antagonistic effects on RNAi-mediated PERV suppression ⁸⁰ .

Gene Editing

Gene editing stands out as an ideal approach for inactivating viruses embedded within the genome. Technologies such as Zinc finger nuclease (ZFN) or clustered regularly interspaced short palindromic repeats–associated RNA-guided DNA endonuclease Cas9 (CRISPR/Cas9) can be harnessed to deactivate the PERV gene ⁸¹ . ZFN technology, designed to target multiple proviral sequences of PERV, exhibited high expression within the nucleus and effective interactions. However, its implementation induced extreme cytotoxicity in PERV-infected cells ⁸² . In contrast, CRISPR/Cas9 has shown greater success. In 2015, Yang and her research team employed CRISPR/Cas9 to eliminate 62 PERV copies in porcine kidney epithelial cell line (PK15), resulting in a reduction of PERV transmission to humans by more than 1,000 times ⁵³ . Subsequently, in 2017, Yang and her team used CRISPR-Cas9 to inactivate all PERV instances in pig primary cell lines and generated PERV-inactivated pigs through somatic cell nuclear transfer ⁴² . Furthermore, in 2021, the team demonstrated that CRISPR-Cas9 and transposon technology could be used to engineer pigs with inactivated PERV, eliminating three xenoantigens and enabling the expression of nine human transgenes. This advancement enhanced the pig’s immune compatibility and coagulation compatibility with humans, successfully addressing the issue of PERV safety in xenotransplantation ⁸³ . However, a notable challenge persists in the form of a highly sensitive method for measuring PERV copy numbers and expression levels^45,53,82. Recent studies have explored the use of cytosine base editors (CBEs), which do not induce DNA double-strand breaks (DSBs) like CRISPR-Cas9, offering a means to edit PERV with reduced cytotoxic effects. In addition, the plasmids employed for PERV editing are not integrated into the host genome, and they do not impact the karyotype of modified cells ⁸⁴ .

Current Status and Prospects for Clinical Trails

Over the past decade, nonhuman primate models have witnessed remarkable advancements, thanks to the emergence of gene editing technologies and refined immunosuppressive regimens. These innovations have propelled research to new heights, as evidenced by documented achievements in xenotransplantation outcomes. Researchers have reported instances of remarkable survival in pig-baboon heterotopic heart xenografts, with some cases extending up to an impressive 945 days, and in orthotopic xenografts, survival periods of up to 195 days have been achieved^85–87. In a groundbreaking development, the University of Maryland School of Medicine disclosed the successful transplantation of gene-edited pig hearts into baboons. These modified pig hearts, with six genes edited, exhibited remarkable resilience, surviving in baboons for a remarkable 264 days with the assistance of life support ⁸⁸ . Furthermore, in a significant milestone in 2022, scientists achieved the transplantation of gene-edited pig hearts into two recently deceased humans. Notably, there were no early signs of rejection observed in either transplanted organ. The transplanted hearts functioned normally with the application of standard posttransplant medications, obviating the need for additional mechanical support ⁸⁹ . These groundbreaking successes underscore the promising prospects of xenotransplantation as a viable solution to address the critical shortage of human donor organs. The successes in kidney xenotransplantation have also been nothing short of impressive, with the longest recorded survival period extending to an astounding 499 days ⁹⁰ . Notably, in 2021, several scientific research teams achieved significant milestones by transplanting gene-edited pig kidneys into two brain-dead human recipients. Remarkably, no instances of hyperacute rejection were observed in these groundbreaking procedures^91,92. In addition, substantial strides have been made in the field of liver xenotransplantation. In a noteworthy development in 2020, 13 gene-edited pig-rhesus monkeys demonstrated remarkable resilience, surviving for 26 days following heterotopic auxiliary liver transplantation ⁹³ . Building on this progress, in 2023, a gene-edited pig-macaque orthotopic auxiliary liver transplant achieved a survival period of 34 days, marking another significant advancement in the field ⁹⁴ . In our center, we successfully conducted multiorgan xenotransplantation procedures in both 2020 and 2022, marking significant milestones in the field of xenotransplantation^95,96. Notably, in 2022, we achieved a remarkable feat by transplanting three organs (liver, kidney, and heart) and three tissues (cornea, skin, and bones) from a 6-gene edited pig into four rhesus monkeys. In these groundbreaking procedures, the heterotopic heart transplant recipients exhibited a survival period of 20 days, while the monkey receiving a combined liver and kidney transplant survived for 14 days ⁹⁶ .

While the successful outcomes of preclinical experiments in recent years have fueled optimism about the progression of xenotransplantation to clinical applications, it is important to note that the Food and Drug Administration (FDA) has not yet granted approval for clinical trials. The world’s first pig heart transplant was conducted under the FDA’s “compassionate use” provision, which allows for experimental treatments when a patient is facing a serious or life-threatening medical condition ⁶ . Using deceased models for xenotransplantation research also poses certain limitations. Studies show hearts procured after brain death manifest distinct pathological changes. The adverse effects of brain death on myocardial function, marked by substantial anaerobic metabolic and hemodynamic decline, trigger a catecholamine storm resulting in heightened tachycardia and hypertension. This escalation drives increased cardiac output and myocardial oxygen consumption, worsening underlying myocardial ischemia, and significantly elevating the risk of postoperative transplant failure^97–100. But xenotransplantation studies from recently deceased donors are critical to gathering the additional human data needed to advance the field. Several crucial steps must be completed before FDA approval can be obtained, including the testing of a clinical immunosuppression model, ensuring the genetic modifications of xenografts are appropriate, and establishing a robust viral surveillance protocol with confirmation of biocompatibility^101–104. Several institutions have announced their intentions to initiate clinical trials in kidney xenotransplantation, with a phase I clinical trial of gene-edited pig-human kidney transplantation registered in 2023, pending U.S. FDA authorization ¹⁰⁵ .

Despite the substantial progress made, the absolute risk of infection in xenotransplantation remains uncertain. Early clinical trials will provide valuable insights into the most suitable monitoring and prophylaxis strategies for xenotransplantation. Given the immunosuppressed state of organ recipients, the anticipation of infections is crucial. Conducting routine pretransplant screenings for recipients will help identify latent infections that necessitate ongoing surveillance or the implementation of prophylactic therapies. Screening source animals for latent and active infections using available assays can help restrict donor-derived infections to a certain extent. Regular monitoring following FDA and other relevant guidance documents involves employing microbe-specific assays. In addition, the implementation of advanced unbiased metagenomic sequencing methods can aid in surveillance for both known and unknown organisms^106,107. Advancements in microbiology, such as quantitative molecular assays for viruses and unbiased metagenomic sequencing, enable the screening and monitoring of recipients and donors for infections, even in the absence of symptoms. But these methods have not yet been validated or approved for clinical use and are known to incur high costs ¹⁰⁸ . Blood samples collected from recipients and contacts can be systematically archived at standard intervals. This archival practice serves the purpose of facilitating future epidemiological studies or advancements in unbiased metagenomic sequencing techniques. Xenotransplantation recipients displaying signs of infection, such as fever, hypotension, or graft dysfunction, may undergo various diagnostic procedures. These include blood, urine, and/or sputum cultures, alongside relevant radiological examinations and invasive diagnostics involving microbiological and histopathological analyses ¹⁰⁹ . Implementing stringent infection control measures is crucial throughout this process. We believe that as clinical data continue to emerge, regulatory, and control guidance on infection will evolve accordingly.

Based on our experiences, we offer the following recommendations for future clinical trials: First, involve virologists in the research design phase to provide valuable insights ⁶² . Second, given the presence of numerous new or unfamiliar viruses in donor animals, the development of more sensitive detection and elimination methods is imperative ²⁶ . Third, considering that the use of immunosuppressants in xenograft recipients can exacerbate infection symptoms, it is essential to assess and optimize the recipient’s condition prior to surgery. Finally, implementing meticulous infection control measures tailored to various groups such as xenotransplant recipients, healthcare workers, contacts, and others is essential.

Conclusions

Recent discoveries have underscored the paramount importance of viral safety in ensuring the success of xenotransplantation. In recent years, significant strides have been made in developing highly sensitive and specific methods for detecting swine viruses, effectively eliminating the majority of known viral threats. Nevertheless, the ongoing emergence of new and latent viruses poses an ongoing challenge to the safety of xenotransplantation, as these viruses carry the potential to infect humans or even trigger fresh outbreaks of disease in pig populations. Looking ahead, our ultimate aspiration is to comprehensively address and resolve the issue of microbiological safety in xenotransplantation. This ambitious goal will require ongoing vigilance, research, and the continuous development of cutting-edge strategies to safeguard the health of both recipients and donor animals.