Animal models of bovine spongiform encephalopathy and vCJD infectivity in blood: two swallows do not a summer make

Studies in animal models have provided extraordinary insight into the pathogenesis of transmissible spongiform encephalopathies (TSEs). Investigators conducting these studies should be praised for their ingenuity and perseverance, and also for their courage in working with such a mysterious and potentially deadly family of infectious agents. They have had to overcome overwhelming barriers to the conduct of such studies, including: the identification of susceptible species for various TSEs; the development of clinical and pathologic criteria and molecular methods to detect and differentiate TSE transmissions; and the requirement for inoculation of a large number of animals in order to obtain quantitative assessments of infectivity. Furthermore, the prolonged incubation periods from exposure to detectable disease necessitate months and even years of observation before experiments can be conclusively interpreted. These investigators are true pioneers. Vostal et al.1 and Brown2 recently reviewed many of the seminal observations derived from these animal TSE studies, with a focus on the expression and physiologic function of normal prion protein in blood cells and the possible pathophysiologic implications of blood cell prions for TSE transmission and pathogenesis. Of particular note to transfusionists are: 1) the studies by Aguzzi and colleagues,3, 4 using transgenic mice and BMT models to define the important role of cellular elements in the immune system (most notably follicular dendritic cells) for trafficking of TSE infectivity from the periphery to the central nervous system (CNS); 2) studies by a number of groups defining the distribution of the normal cellular prion conformer (PrPc) and the infectious, proteinase-resistant form (PrPres) within the cellular and plasma compartments of blood;5-8 and 3) studies using susceptible animals for quantitative assays for TSE infectivity to determine the reduction in infectivity during fractionation procedures used to prepare therapeutic plasma derivatives.9-11 Many animal model studies reveal that the pathogenesis of TSEs varies substantially among different host species. This is true even when different species are inoculated with infectious materials from the same disease. Further, experimental infections may differ in important ways from natural disease. These differences may include the levels of TSE agent required to infect, the routes by which infection can be transmitted, the clinical and pathologic manifestations of infection and the incubation periods prior to disease. As documented in this issue of TRANSFUSION by Barclay et al.,12 PrPc expression on blood cells and in plasma differs substantially from species to species. Barclay et al. studied the distribution of PrPc (as a surrogate of PrPres) using an optimized flow cytometric assay and a series of anti-PrPc MoAbs and rabbit polyclonal antisera. They examined major blood cells from healthy humans, mice, hamsters, cattle, and sheep. They documented dramatic differences in prion reactivity on blood cells with different antibodies and in different species. Depending on the assay and the species, PrPc can be found on virtually any combination of RBCs, platelets and most WBC subsets (excluding eosinophils), as well as in plasma. The authors raise cautions over using animal studies of prion distribution to predict infectivity in blood during natural and experimental TSE infections of animals, let alone humans. This issue of TRANSFUSION also contains an article by Simak et al.,13 demonstrating that PrPc is expressed at high levels on cultured human and bovine endothelial cells. These investigators use multiple methods to confirm this finding, including flow cytometry, reverse transcriptase PCR, and Western blot assays. These observations and others14, 15 suggest that a substantial proportion of the PrPc in blood is not cell-associated, but rather is suspended in plasma as microvesicles or particulate forms released from endothelium, platelets or RBCs. Of note, neither infectivity nor PrPc in the plasma compartment may escape removal by WBC-reduction filters.5, 15 An issue of critical interest and importance to the readers of TRANSFUSION is the degree to which TSEs may be transmitted from affected, presymptomatic individuals via blood transfusion. Clearly, there are but two available options to explore this question: epidemiologic studies with humans, and examination of appropriate animal models. As discussed several years ago, neither approach can ensure the absence of such transmission, although it is reasonable to suppose that epidemiologic studies might at least provide data about the limits of risk. Therefore, continued research involving both animal model studies and aggressive epidemiologic surveillance is critical. Although there are only a small number of published reports relating to infectivity models of transfusion-transmitted TSEs,1, 2 a number of studies have recently been conducted or are now in progress. These studies use TSEs adapted into small animal model systems. They tend to fall into two broad categories: those that demonstrate the presence of infectivity in the blood through the use of intracranial inoculation into indicator animals, and those that use IV inoculation as a stricter model of transfusion. In rodent models based on classical TSE inocula, and more recently rodent-adapted bovine spongiform encephalopathy (BSE) and vCJD, it has been conclusively shown that infectivity is present in the blood during and prior to symptomatic disease.5, 16, 17 Such infectivity may be transmitted (albeit with lower efficiency) via the IV route. Interestingly, there is essentially no evidence of transmission of infection by IV inoculation of blood taken directly from animals or humans with spontaneous or naturally acquired TSE disease, despite attempts to do so with scrapie-infected sheep, BSE-infected cattle, and vCJD-infected human blood. One consequence of the heterogeneity in the species-specific distribution of prions on blood cells and the pathogenicity of TSEs has been the recognition that work on blood transmission of classical TSEs in animal models may not be applicable to transfusion of blood from humans with TSEs. In particular, the documentation of infectivity in animal models of classical CJD is not consistent with the increasing body of epidemiologic data, which fails to show any evidence of transfusion-related transmission of classical CJD. These studies include statistical analyses of the prevalence of classical CJD cases among the general population vs. recipients of blood and derivatives, lookback investigations of the recipients of transfusions known to have been donated by persons who later died of CJD, and intensive surveillance (including brain pathology) of high-risk groups such as patients with hemophilia or congenital bleeding disorders.18-23 As pointed out earlier, it is not possible to be confident in extending any of these studies from classical TSEs to BSE or, more particularly, to the human form, vCJD. While it is clear that the pathogenesis of this disease varies in important ways from that of classical TSEs, there are insufficient data to define the overall impact of these differences. Additionally, the extent of the epidemic of human infection with the BSE agent is unknown. It is perhaps encouraging that estimates of the eventual number of cases are declining, with two recent reports projecting a range of 205 to several thousand total cases.24, 25 However, such estimates are critically dependent on the nonquantifiable assumptions that underlie them. Regarding the direct question of vCJD transmission by transfusion, active surveillance has established that eight of the 114 cases reported in Great Britain through January 2002 had donated blood within the year preceding diagnosis; none of the 22 recipients of labile blood components from these donations have developed disease.26 In addition, four of the vCJD cases had been transfused prior to their diagnosis; all of the donors of these transfused units are well, and this rate of transfusion history is expected given the age distribution of vCJD cases.26 How then should we interpret two recent studies, one in the current issue of TRANSFUSION, relating to the transmission of the agent of BSE via blood? This question is of more than passing interest, because laboratory-derived information about TSEs, particularly about BSE, continues to have a disproportionate impact on the development of blood safety policy. Approximately 2 years ago, Houston et al.27 described the transmission of the BSE agent from an orally infected, but asymptomatic sheep, by transfusion of some 400 mL of blood to another sheep. In an accompanying editorial, Paul Brown, MD, cautioned care in interpretation of this single-animal study and even asked whether the information should have been published in such a preliminary form.28 Should we not ask similar questions about the paper by Bons et al.29 in this issue of TRANSFUSION, particularly as Brown is himself a coauthor? Bons et al. report on the transmission of the BSE agent in a prosimian (lemur) model. The agent was derived from an experimentally infected cynomolgus monkey, passaged once into a lemur which, once symptomatic, served as the donor. Two recipient animals were inoculated intracranially and intraperitoneally with brain tissue and one was inoculated intracranially with buffy coat. All three recipient animals developed symptomatic and histologically proven TSE disease after the same incubation period. The authors tentatively conclude that blood-borne transmission has been demonstrated in a primate model, for the first time, even though the infection was demonstrated by intracranial inoculation. We heartily endorse the cautions expressed in the authors' own discussion. But we would additionally question the relevance of the lemur model as truly representative of primates as a whole, and note again that the blood-borne observation applies to a single animal and that intracranial rather than IV inoculation of buffy coat was used to demonstrate infectivity. No person or institution would want to be in the position of allowing the transmission of vCJD (or indeed, any other TSE) by transfusion. Thus, extremely conservative policies have been developed in the area of donor and product management in the face of the potential risk for vCJD infection. These policies are generally compatible with the precautionary principle, although they seem to go well beyond commentary relating to the appropriate implementation of this principle.30 This may be understandable in the context of the known severity of vCJD and the absence of detailed knowledge about the size of the epidemic and the properties of the agent. However, we are very concerned about the extent to which observations from animal models that are not substantiated by epidemiologic findings provoke inappropriate or premature policy decisions. For example, Aguzzi's exquisite observations on the involvement of the immune system in trafficking of TSE infection to the CNS following peripheral inoculation led to a cascade of countries implementing nationwide WBC reduction programs. The rationale was based on early studies suggesting a direct role for B cells in the dissemination of PrPc within the body.3 Yet further studies suggested that the effect was ultimately attributable to endogenous antigen-presenting cells, and that WBC reduction would have a limited effect on blood TSE content.4, 5, 15 The preliminary observation (still unconfirmed) by Houston et al.27 of transfusion transmission of BSE in sheep led directly to the hasty implementation of a BSE-related travel deferral in Australia (Albert Farrugia, oral communication, January 2002). With these events as background, we wonder to what the Bons et al. report29 might lead. Most readers of TRANSFUSION support evidence-based medicine, and the incorporation of science into policy development. Preliminary scientific observations from animal models should not serve as a basis for policy. Even excellent experimental science may be but the first step in an evolving truth. In terms of public health policy, it is critical that decisions be grounded by clinical and epidemiologic studies. Undoubtedly the final answer to the question of whether vCJD is transmissible by transfusion will come from Great Britain, where it seems likely that millions of blood components from donors exposed to BSE have been transfused since the tragic epidemic began. We, as authors, reviewers, and readers, must be conscious of the need to gauge the impact and value of any publication. Furthermore, we must be willing to stand up and counsel others not only about the value of science, but also about its limitations.