Long-range inhibition synchronizes and updates prefrontal task activity.

Changes in patterns of activity within the medial prefrontal cortex enable rodents, non-human primates and humans to update their behaviour to adapt to changes in the environment-for example, during cognitive tasks1-5. Parvalbumin-expressing inhibitory neurons in the medial prefrontal cortex are important for learning new strategies during a rule-shift task6-8, but the circuit interactions that switch prefrontal network dynamics from maintaining to updating task-related patterns of activity remain unknown. Here we describe a mechanism that links parvalbumin-expressing neurons, a new callosal inhibitory connection, and changes in task representations. Whereas nonspecifically inhibiting all callosal projections does not prevent mice from learning rule shifts or disrupt the evolution of activity patterns, selectively inhibiting only callosal projections of parvalbumin-expressing neurons impairs rule-shift learning, desynchronizes the gamma-frequency activity that is necessary for learning8 and suppresses the reorganization of prefrontal activity patterns that normally accompanies rule-shift learning. This dissociation reveals how callosal parvalbumin-expressing projections switch the operating mode of prefrontal circuits from maintenance to updating by transmitting gamma synchrony and gating the ability of other callosal inputs to maintain previously established neural representations. Thus, callosal projections originating from parvalbumin-expressing neurons represent a key circuit locus for understanding and correcting the deficits in behavioural flexibility and gamma synchrony that have been implicated in schizophrenia and related conditions9,10.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Recommendations for procurement of starting materials by apheresis for advanced therapy medicinal products.

Activities involved in the production of certain advanced therapy medicinal products (ATMPs) require standardized approaches to mononuclear cell procurement to ensure the highest product quality, safety and process efficiency. These aims must be achieved while meeting regulatory and accreditation requirements for the procurement of mononuclear cells as starting materials. Mononuclear cells constitute the starting materials for many ATMPs, and this article sets out recommendations for procurement by clinical apheresis, addressing the variation among existing working practices and different manufacturers' requirements that currently poses a challenge when managing multiple different protocols.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

Estimation of manufacturing development costs of cell-based therapies: a feasibility study.

BACKGROUND AIMS: Cell-based therapies (CBTs) provide opportunities to treat rare and high-burden diseases. Manufacturing development of these innovative products is said to be complex and costly. However, little research is available providing insight into resource use and cost drivers. Therefore, this study aimed to assess the feasibility of estimating the cost of manufacturing development of two cell-based therapy case studies using a CBT cost framework specifically designed for small-scale cell-based therapies. METHODS: A retrospective costing study was conducted in which the cost of developing an adoptive immunotherapy of Epstein-Barr virus-specific cytotoxic T lymphocytes (CTLs) and a pluripotent stem cell (PSC) master cell bank was estimated. Manufacturing development was defined as products advancing from technology readiness level 3 to 6. The study was conducted in a Scottish facility. Development steps were recreated via developer focus groups. Data were collected from facility administrative and financial records and developer interviews. RESULTS: Application of the manufacturing cost framework to retrospectively estimate the manufacturing design cost of two case studies in one Scottish facility appeared feasible. Manufacturing development cost was estimated at £1,201,016 for CTLs and £494,456 for PSCs. Most costs were accrued in the facility domain (56% and 51%), followed by personnel (20% and 32%), materials (19% and 15%) and equipment (4% and 2%). CONCLUSIONS: Based on this study, it seems feasible to retrospectively estimate resources consumed in manufacturing development of cell-based therapies. This fosters inclusion of cost in the formulation and dissemination of best practices to facilitate early and sustainable patient access and inform future cost-conscious manufacturing design decisions.

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017.

This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. Stem Cells 2019;37:1130-1135.

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.

BACKGROUND AIMS: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies. METHODS: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€). RESULTS: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices. CONCLUSIONS: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs.

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.

Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552-1566.

Development, functional characterization and validation of methodology for GMP-compliant manufacture of phagocytic macrophages: A novel cellular therapeutic for liver cirrhosis.

BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial). METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product. RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest. CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

A prion reduction filter does not completely remove endogenous prion infectivity from sheep blood.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy affecting humans, acquired initially through infection with bovine spongiform encephalopathy (BSE). A small number of vCJD cases have been acquired through the transfusion of blood from asymptomatic donors who subsequently developed vCJD. Filter devices that selectively bind the infectious agent associated with prion disease have been developed for removal of infection from blood. This study independently assessed one such filter, the P-CAPT filter, for efficacy in removing infectivity associated with the BSE agent in sheep blood. The sheep BSE model has previously been used to evaluate the distribution of infectivity in clinically relevant blood components. This is the first study to assess the ability of the P-CAPT filter to remove endogenous infectivity associated with blood components prepared from a large animal model. STUDY DESIGN AND METHODS: Paired units of leukoreduced red blood cells (LR-RBCs) were prepared from donors at the clinical stage of infection and confirmed as having BSE. One cohort of recipients was transfused with LR-RBCs alone, whereas a parallel cohort received LR and P-CAPT-filtered RBCs (LR-RBCs-P-CAPT). RESULTS: Of 14 recipients, two have been confirmed as having BSE. These sheep had received LR-RBCs and LR-RBCs-P-CAPT from the same donor. CONCLUSIONS: The results indicate that, after leukoreduction and P-CAPT filtration, there can still be sufficient residual infectivity in sheep RBCs to transmit infection when transfused into a susceptible recipient.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease.

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow.

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte-colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34(+)CD133(-)CD146(+) cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real-time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow-derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34(+)CD133(-)CD146(+) mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow-derived circulating precursor for endothelial cells.

A randomized controlled trial of peripheral blood mononuclear cell depletion in experimental human lung inflammation.

RATIONALE: Depletion of monocytes reduces LPS-induced lung inflammation in mice, suggesting monocytes as potential therapeutic targets in acute lung injury. OBJECTIVES: To investigate whether depletion of circulating blood monocytes has beneficial effects on markers of systemic and pulmonary inflammation in a human model of acute lung inflammation. METHODS: A total of 30 healthy volunteers were enrolled in a randomized controlled trial. Volunteers inhaled LPS at baseline, and were randomized to receive active mononuclear cell depletion by leukapheresis, or sham leukapheresis, in a double-blind fashion (15 volunteers per group). Serial blood counts were measured, bronchoalveolar lavage (BAL) was performed at 9 hours, and [(18)F]fluorodeoxyglucose positron emission tomography at 24 hours. The primary endpoint was the increment in circulating neutrophils at 8 hours. MEASUREMENTS AND MAIN RESULTS: As expected, inhalation of LPS induced neutrophilia and an up-regulation of inflammatory mediators in the blood and lungs of all volunteers. There was no significant difference between the depletion and sham groups in the mean increment in blood neutrophil count at 8 hours (6.16 × 10(9)/L and 6.15 × 10(9)/L, respectively; P = 1.00). Furthermore, there were no significant differences in BAL neutrophils or protein, positron emission tomography-derived measures of global lung inflammation, or cytokine levels in plasma or BAL supernatant between the study groups. No serious adverse events occurred, and no symptoms were significantly different between the groups. CONCLUSIONS: These findings do not support a role for circulating human monocytes in the early recruitment of neutrophils during LPS-mediated acute lung inflammation in humans.

Sensitivity to biases of case-control studies on medical procedures, particularly surgery and blood transfusion, and risk of Creutzfeldt-Jakob disease.

BACKGROUND: Evidence of risk of Creutzfeldt-Jakob disease (CJD) associated with medical procedures, including surgery and blood transfusion, is limited by susceptibility to bias in epidemiological studies. METHODS: Sensitivity to bias was explored using a central-birth-cohort model using data from 18 case-control studies obtained after a review of 494 reports on medical procedures and risk of CJD, systematic for the period January 1, 1989 to December 31, 2011. RESULTS: The validity of the findings in these studies may have been undermined by: recall; control selection; exposure assessment in life-time periods of different duration, out of time-at-risk of effect, or asymmetry in case/control data; and confounding by concomitant blood transfusion at the time of surgery. For sporadic CJD (sCJD), a history of surgery or blood transfusion was associated with risk in some, but not all, recent studies at a ≥10 year lag time, when controls were longitudinally sampled. Space-time aggregation of surgical events was not seen. Surgery at early clinical onset might be overrepresented among cases. Neither surgical history nor blood transfusion unlabelled for donor status, dental treatments or endoscopic examinations were linked to variant CJD (vCJD). CONCLUSIONS: These results indicate the need for further research. Common challenges within these studies include access to and content of past medical/dental treatment records for diseases with long incubation periods.

Human embryonic stem cells rapidly take up and then clear exogenous human and animal prions in vitro.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host-encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self-renewing populations. Following undirected differentiation, up-regulation of prion protein expression occurred in each of the major embryonic lineages. Self-renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease-associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation.

Subclinical infection occurs frequently following low dose exposure to prions by blood transfusion.

Infectious prion diseases have very long incubation periods, and the role that subclinical infections play in transmission, persistence and re-emergence of these diseases is unclear. In this study, we used a well-established model of vCJD (sheep experimentally infected with bovine spongiform encephalopathy, BSE) to determine the prevalence of subclinical infection following exposure by blood transfusion from infected donors. Many recipient sheep survived for years post-transfusion with no clinical signs and no disease-associated PrP (PrPSc) found in post mortem tissue samples by conventional tests. Using a sensitive protein misfolding cyclic amplification assay (PMCA), we found that the majority of these sheep had detectable PrPSc in lymph node samples, at levels approximately 105-106 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrPSc in other tissues, including brain, but not in blood samples. The results demonstrate that subclinical infection is a frequent outcome of low dose prion infection by a clinically relevant route for humans (blood transfusion). The long term persistence of low levels of infection has important implications for prion disease control and the risks of re-emergent infections in both humans and animals.