Infectious Diseases and Clinical Xenotransplantation.

Xenotransplantation, transplantation into humans of vascularized organs or viable cells from nonhuman species, is a potential solution to shortages of transplantable human organs. Among challenges to application of clinical xenotransplantation are unknown risks of transmission of animal microbes to immunosuppressed recipients or the community. Experience in allotransplantation and in preclinical models suggests that viral infections are the greatest concern. Worldwide, the distribution of swine pathogens is heterogeneous and cannot be fully controlled by international agricultural regulations. It is possible to screen source animals for potential human pathogens before procuring organs in a manner not possible within the time available for surveillance testing in allotransplantation. Infection control measures require microbiological assays for surveillance of source animals and xenograft recipients and research into zoonotic potential of porcine organisms. Available data suggest that infectious risks of xenotransplantation are manageable and that clinical trials can advance with appropriate protocols for microbiological monitoring of source animals and recipients.

Research opportunities and ethical considerations for heart and lung xenotransplantation research: A report from the National Heart, Lung, and Blood Institute workshop.

Xenotransplantation offers the potential to meet the critical need for heart and lung transplantation presently constrained by the current human donor organ supply. Much was learned over the past decades regarding gene editing to prevent the immune activation and inflammation that cause early organ injury, and strategies for maintenance of immunosuppression to promote longer-term xenograft survival. However, many scientific questions remain regarding further requirements for genetic modification of donor organs, appropriate contexts for xenotransplantation research (including nonhuman primates, recently deceased humans, and living human recipients), and risk of xenozoonotic disease transmission. Related ethical questions include the appropriate selection of clinical trial participants, challenges with obtaining informed consent, animal rights and welfare considerations, and cost. Research involving recently deceased humans has also emerged as a potentially novel way to understand how xeno-organs will impact the human body. Clinical xenotransplantation and research involving decedents also raise ethical questions and will require consensus regarding regulatory oversight and protocol review. These considerations and the related opportunities for xenotransplantation research were discussed in a workshop sponsored by the National Heart, Lung, and Blood Institute, and are summarized in this meeting report.

Adjunctive glucocorticoid therapy for Pneumocystis jirovecii pneumonia in solid organ transplant recipients: A multicenter cohort, 2015-2020.

Solid organ transplant recipients (SOTRs) frequently receive adjunctive glucocorticoid therapy (AGT) for Pneumocystis jirovecii pneumonia (PJP). This multicenter cohort of SOTRs with PJP admitted to 20 transplant centers in Canada, the United States, Europe, and Australia, was examined for whether AGT was associated with a lower rate of all-cause intensive care unit (ICU) admission, 90-day death, or a composite outcome (ICU admission or death). Of 172 SOTRs with PJP (median [IQR] age: 60 (51.5-67.0) years; 58 female [33.7%]), the ICU admission and death rates were 43.4%, and 20.8%, respectively. AGT was not associated with a reduced risk of ICU admission (adjusted odds ratio [aOR] [95% CI]: 0.49 [0.21-1.12]), death (aOR [95% CI]: 0.80 [0.30-2.17]), or the composite outcome (aOR [95% CI]: 0.97 [0.71-1.31]) in the propensity score-adjusted analysis. AGT was not significantly associated with at least 1 unit of the respiratory portion of the Sequential Organ Failure Assessment score improvement by day 5 (12/37 [32.4%] vs 39/111 [35.1%]; P = .78). We did not observe significant associations between AGT and ICU admission or death in SOTRs with PJP. Our findings should prompt a reevaluation of routine AGT administration in posttransplant PJP treatment and highlight the need for interventional studies.

Heart of the matter-infection and xenotransplantation.

In this clinicopathological conference, invited experts discussed a previously published case of a patient with nonischemic cardiomyopathy who underwent heart transplantation from a genetically modified pig source animal. His complex course included detection of porcine cytomegalovirus by plasma microbial cell-free DNA and eventual xenograft failure. The objectives of the session included discussion of selection of immunosuppressive regimens and prophylactic antimicrobials for human xenograft recipients, description of infectious disease risk assessment and mitigation in potential xenograft donors and understanding of screening and therapeutic strategies for potential xenograft-related infections.

Infection and clinical xenotransplantation: Guidance from the Infectious Disease Community of Practice of the American Society of Transplantation.

This guidance was developed to summarize current approaches to the potential transmission of swine-derived organisms to xenograft recipients, health care providers, or the public in clinical xenotransplantation. Limited specific data are available on the zoonotic potential of pig pathogens. It is anticipated that the risk of zoonotic infection in xenograft recipients will be determined by organisms present in source animals and relate to the nature and intensity of the immunosuppression used to maintain xenograft function. Based on experience in allotransplantation and with preclinical models, viral infections are of greatest concern, including porcine cytomegalovirus, porcine lymphotropic herpesvirus, and porcine endogenous retroviruses. Sensitive and specific microbiological assays are required for routine microbiological surveillance of source animals and xenograft recipients. Archiving of blood samples from recipients, contacts, and hospital staff may provide a basis for microbiological investigations if infectious syndromes develop. Carefully implemented infection control practices are required to prevent zoonotic pathogen exposures by clinical care providers. Informed consent practices for recipients and their close contacts must convey the lack of specific data for infectious risk assessment. Available data suggest that infectious risks of xenotransplantation are manageable and that clinical trials can advance with carefully developed protocols for pretransplant assessment, syndrome evaluation, and microbiological monitoring.

American Society of Transplant Surgeons-American Society of Transplantation report of FDA meeting on regulatory expectations for xenotransplantation products.

In June 2022, the US Food and Drug Administration Center for Biologics Evaluation and Research held the 73rd meeting of the Cellular, Tissue, and Gene Therapies Advisory Committee for public discussion of regulatory expectations for xenotransplantation products. The members of a joint American Society of Transplant Surgeons/American Society of Transplantation committee on xenotransplantation compiled a meeting summary focusing on 7 topics believed to be key by the committee: (1) preclinical evidence supporting progression to a clinical trial, (2) porcine kidney function, (3) ethical aspects, (4) design of initial clinical trials, (5) infectious disease issues, (6) industry perspectives, and (7) regulatory oversight.

PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Expert opinion on the identification, risk assessment, and mitigation of microorganisms and parasites relevant to xenotransplantation products from pigs.

Xenotransplantation has the potential to address shortages of organs available for clinical transplantation, but concerns exist regarding potential risks posed by porcine microorganisms and parasites (MP) to the health of human recipients. In this study, a risk-based framework was developed, and expert opinion was elicited to evaluate porcine MP based on swine exposure and risk to human health. Experts identified 255 MP to include in the risk assessment. These were rated by experts for five criteria regarding potential swine exposure in the USA and human health risks. MP were subsequently categorized into three risk mitigation groups according to pre-defined rules: disqualifying porcine MP (due to their pathogenic potential, n = 130); non-disqualifying porcine MP (still relevant to consider for biosecurity or monitoring efforts, n = 40); and alert/watch list (not reported in the USA or MP not in swine, n = 85). Most disqualifying (n = 126) and non-disqualifying (n = 36) porcine MP can effectively be eliminated with high biosecurity programs. This approach supports surveillance and risk mitigation strategies for porcine MP in swine produced for xenotransplantation, such as documentation of freedom from porcine MP, or use of porcine MP screening, monitoring, or elimination options. To the authors' knowledge, this is the first effort to comprehensively identify all relevant porcine MP systematically and transparently evaluate the risk of infection of both donor animals and immunosuppressed human recipients, and the potential health impacts for immunosuppressed human recipients from infected xenotransplantation products from pigs.

Moving xenotransplantation from bench to bedside: Managing infectious risk.

Xenotransplantation of organs from swine in immunosuppressed human recipients poses many of the same challenges of allotransplantation relative to the risk for infection, malignancy, or graft rejection in proportion to the degree of immunosuppression and epidemiologic exposures. The unique features of xenotransplantation from pigs relative to infectious risk center on the potential for unusual organisms derived from swine causing productive infection, "xenosis" or "xenozoonosis," in the host. Based on experience in allotransplantation, the greatest hazard is due to viruses, due to the relative lack of information regarding the behavior of these potential pathogens in humans, the absence of validated serologic and molecular assays for swine-derived pathogens, and uncertainty regarding the efficacy of therapeutic agents for these organisms. Other known, potential pathogens (i.e., bacteria, fungi, parasites) tend to be comparable to those of humans. Concerns remain for unknown organisms in swine that may replicate in immunosuppressed humans. Clinical trials of genetically modified organs sourced from swine in immunosuppressed humans with organ failure are under development. Such trials require informed consent regarding potential infectious risks to the recipient, determination of breeding characteristics of swine, assessments of potential risks to the public and healthcare providers, consideration of ethical issues posed by this novel therapy, and defined strategies to monitor and address infectious episodes that may be encountered by healthcare teams. Clinical trials in xenotransplantation will allow improved definition of potential infectious risks.

Progress Toward Cardiac Xenotransplantation.

Consistent survival of life-supporting pig heart xenograft recipients beyond 90 days was recently reported using genetically modified pigs and a clinically applicable drug treatment regimen. If this remarkable achievement proves reproducible, published benchmarks for clinical translation of cardiac xenografts appear to be within reach. Key mechanistic insights are summarized here that informed recent pig design and therapeutic choices, which together appear likely to enable early clinical translation.

Immunosuppressive Agents and Infectious Risk in Transplantation: Managing the "Net State of Immunosuppression".

Successful solid organ transplantation reflects meticulous attention to the details of immunosuppression, balancing risks for graft rejection against risks for infection. The "net state of immune suppression" is a conceptual framework of all factors contributing to infectious risk. Assays that measure immune function in the immunosuppressed transplant recipient relative to infectious risk and allograft function are lacking. The best measures of integrated immune function may be quantitative viral loads to assess the individual's ability to control latent viral infections. Few studies address adjustment of immunosuppression during active infections; thus, confronted with infection in solid organ recipients, the management of immunosuppression is based largely on clinical experience. This review examines known measures of immune function and the immunologic effects of common immunosuppressive drugs and available studies reporting modification of drug regimens for specific infections. These data provide a conceptual framework for the management of immunosuppression during infection in organ recipients.

Haploidentical hematopoietic cell and kidney transplantation for hematological malignancies and end-stage renal failure.

At Massachusetts General Hospital, we pioneered simultaneous hematopoietic cell (HCT)/kidney transplantation from HLA-identical related donors for the treatment of hematological malignancies with end-stage renal failure. We have now extended this to HLA-haploidentical donors in a pilot trial. Six recipients, 5 of whom were conditioned with fludarabine, cyclophosphamide, and total-body irradiation, underwent combined HCT/kidney transplantation from haploidentical donors; graft-versus-host disease (GVHD) prophylaxis included post-HCT cyclophosphamide, tacrolimus, and mycophenolate mofetil. One patient died as a result of complications of fludarabine neurological toxicity. No neurological toxicity was observed in subsequent patients who received lower fludarabine doses and more intense postfludarabine dialysis. There were no cases of grade 2 to 4 acute GVHD and 1 case of moderate chronic GVHD by 12 months. One patient experienced relapse of multiple myeloma at 30 months after HCT and died 4 years posttransplantation. Overall, 4 of 6 patients remain alive, without disease relapse and with long-term renal rejection-free survival. This trial was registered at www.clinicaltrials.gov as #NCT01758042.

Early detection of SARS-CoV-2 and other infections in solid organ transplant recipients and household members using wearable devices.

The increasing global prevalence of SARS-CoV-2 and the resulting COVID-19 disease pandemic pose significant concerns for clinical management of solid organ transplant recipients (SOTR). Wearable devices that can measure physiologic changes in biometrics including heart rate, heart rate variability, body temperature, respiratory, activity (such as steps taken per day) and sleep patterns, and blood oxygen saturation show utility for the early detection of infection before clinical presentation of symptoms. Recent algorithms developed using preliminary wearable datasets show that SARS-CoV-2 is detectable before clinical symptoms in >80% of adults. Early detection of SARS-CoV-2, influenza, and other pathogens in SOTR, and their household members, could facilitate early interventions such as self-isolation and early clinical management of relevant infection(s). Ongoing studies testing the utility of wearable devices such as smartwatches for early detection of SARS-CoV-2 and other infections in the general population are reviewed here, along with the practical challenges to implementing these processes at scale in pediatric and adult SOTR, and their household members. The resources and logistics, including transplant-specific analyses pipelines to account for confounders such as polypharmacy and comorbidities, required in studies of pediatric and adult SOTR for the robust early detection of SARS-CoV-2, and other infections are also reviewed.

Prevention of infection in xenotransplantation: Designated pathogen-free swine in the safety equation.

Post-transplantation infections are common. In immunosuppressed human xenograft recipients, infection is most likely to be due to the same pathogens seen in human allotransplantation. However, organisms derived from swine and transmitted with xenografts have the potential to cause novel infections in xenograft recipients. The specific organisms likely to cause infection or "xenosis" are unknown but are postulated to be like those causing infection in allograft recipients. On this basis, theoretical exclusion criteria have been developed to guide the development of source animal herds. Herds developed based on the exclusion of potential human pathogens have been termed "designated pathogen-free" (DPF). Lists of potential pathogens will require revision with changing epidemiology of infection in swine worldwide and clinical experience. Development of new microbiological assays is required both for animal screening and in clinical diagnosis should infections occur. Genetic modifications of swine have the potential to eliminate certain infectious agents such as the porcine endogenous retrovirus; infectious complications of such modifications have not been observed. Unexpected, off target effects of genetic modifications require further study. Monitoring for infection in asymptomatic recipients is important to define infectious risks which are unknown in the absence of clinical trials data. Advanced microbiological techniques may be applied to diagnose and prevent infection in xenograft recipients.

COVID-19 in solid organ transplant recipients: Dynamics of disease progression and inflammatory markers in ICU and non-ICU admitted patients.

BACKGROUND: COVID-19 infection varies in severity from minimal symptoms to critical illness associated with a hyperinflammatory response. Data on disease progression in immunosuppressed solid organ transplant (SOT) recipients are limited. METHODS: We examined the electronic medical records of all SOT recipients with COVID-19 from 12 Massachusetts hospitals between February 1, and May 6, 2020. We analyzed the demographics, clinical parameters, course, and outcomes of illness in these patients. RESULTS: Of 52 COVID-19-positive SOT patients, 77% were hospitalized and 35% required ICU admission. Sixty-nine percent of hospitalized patients had immunosuppression reduced, 6% developed suspected rejection. Co-infections occurred in 45% in ICU vs 5% in non-ICU patients (P = .037). A biphasic pattern of evolution of laboratory tests was observed. In the first 5 days of illness, inflammatory markers were moderately increased. Subsequently, WBC, CRP, ferritin, and D Dimer increased with increasing stay in the ICU, and lymphocyte counts were similar. Five patients (16%) died. CONCLUSIONS: Our data indicate that SOT is associated with high rate of hospitalization, ICU admission, and death from COVID-19 compared to data in the general population of patients with COVID-19. Despite reduction in immunosuppression, suspected rejection was rare. The clinical course and trend of laboratory biomarkers is biphasic with a later, pronounced peak in inflammatory markers seen in those admitted to an ICU. CRP is a useful marker to monitor disease progression in SOT.

Pneumocystis jiroveci.

Pneumocystis jiroveci remains an important fungal pathogen in a broad range of immunocompromised hosts. The natural reservoir of infection remains unknown. Pneumocystis jiroveci Pneumonia (PJP) develops via airborne transmission or reactivation of inadequately treated infection. Nosocomial clusters of infection have been described among immunocompromised hosts. Subclinical infection or colonization may occur. Pneumocystis pneumonia occurs most often within 6 months of organ transplantation and with intensified or prolonged immunosuppression, notably with corticosteroids. Infection is also common during neutropenia and low-lymphocyte counts, with hypogammaglobulinemia, and following cytomegalovirus (CMV) infection. The clinical presentation generally includes fever, dyspnea with hypoxemia, and nonproductive cough. Chest radiographic patterns are best visualized by computed tomography (CT) scan with diffuse interstitial processes. Laboratory examination reveals hypoxemia, elevated serum lactic dehydrogenase levels, and elevated serum (1→3) ß-D-glucan assays. Specific diagnosis is achieved using respiratory specimens with direct immunofluorescent staining; invasive procedures may be required and are important to avoid unnecessary therapies. Quantitative nucleic acid amplification is a useful adjunct to diagnosis but may be overly sensitive. Trimethoprim-sulfamethoxazole (TMP-SMX) remains the drug of choice for therapy; drug allergy should be documented before resorting to alternative therapies. Adjunctive corticosteroids may be useful early in the clinical course; aggressive reductions in immunosuppression may provoke immune reconstitution syndromes. Pneumocystis pneumonia (PJP) prophylaxis is recommended and effective for immunocompromised individuals in the most commonly affected risk groups.

Inflammatory and Infectious Syndromes Associated With Cancer Immunotherapies.

Immunotherapy using antibodies to immune checkpoint molecules or targeted chimeric antigen receptor-modified T cells (CAR-T cells) represent dramatic advances in cancer treatment. These therapies mediate immune-related adverse events that may mimic or amplify infectious presentations. Checkpoint inhibitor therapy may be associated with diverse irAEs including mild skin, endocrine, and autoimmune manifestations or severe inflammatory processes including colitis, pneumonitis, myocarditis, and shock. CAR-T-cell therapies may induce toxicities including cytokine-release syndrome with fevers and multiorgan dysfunction, CAR-T-cell-related encephalopathy syndrome with altered mental status and neurologic dysfunction, or hemophagocytic lymphohistiocytosis-macrophage-activation syndrome. Infectious risks may relate to prior cancer therapies or to treatments of inflammatory dysregulation, including corticosteroids and inhibitors of tumor necrosis factor-α and interleukin-6. Immune activation may unmask subclinical infections. Clinical approaches must attempt to identify infections in the face of immunotherapy-associated inflammatory processes. Empirical antimicrobial therapies should not be delayed based on the presumption of noninfectious syndromes.

Development of transplantable B-cell lymphomas in the MHC-defined miniature swine model.

BACKGROUND: Establishment of transplantable tumors in clinically relevant large animals allows translational studies of novel cancer therapeutics. METHODS: Here we describe the establishment, characterization, and serial transplantation of a naturally occurring B-cell lymphoma derived from a unique, highly inbred sub-line of Massachusetts General Hospital (MGH) major histocompatibility complex (MHC)-defined miniature swine. RESULTS: The lymphoblastic cell line (LCL) originated from peripheral blood of a 2.5 year old female swine leukocyte antigen (SLA)dd-inbred miniature swine breeder demonstrating clinical signs of malignancy. Flow cytometric phenotypic analysis of subclones derived from the original cell line revealed surface markers commonly expressed in a B-cell lineage neoplasm. A subclone of the original LCL was transplanted into mildly-conditioned histocompatible miniature swine and immunocompromised NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Tissue and blood samples harvested 2 weeks following subcutaneous and intravenous injection in a highly inbred SLAdd pig were cultured for tumor growth and phenotypic analysis before serial transfer into NSG mice. Evidence of tumor growth in vivo was found in all tumor cell recipients. In vitro growth characteristics and surface phenotype were comparable between the original and serially transplanted tumor cell lines. CONCLUSIONS: These results indicate the feasibility of developing a large-animal transplantable tumor model using cells derived from spontaneously occurring hematologic malignancies within the highly inbred miniature swine herd.