Chapter 2.9
Human Acquired Immunity to Rotavirus Disease and Correlates of Protection
D. Herrera
M. Parra
M.A. Franco
J. Angel Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Bogotá, Colombia
Abstract
This chapter reviews recent studies from this group on adaptive immune responses to rotavirus (RV) and correlates of protection against disease. The main findings are that both circulating memory RV-specific B and T cells preferentially express intestinal homing receptors, and that both types of lymphocyte populations are enriched in cells with peculiar functions: while B cells are enriched in an innate immunity subset, T cells have a poor functional profile. These characteristics are probably due to the fact that these lymphocytes are activated and proliferate in a tolerogenic intestinal microenvironment (the predominant site of RV replication), making RV infection an excellent model to study lymphocytes from the gut-associated lymphoid tissue. This knowledge may also be useful to define better correlates of protection against the disease.
Keywords
rotavirus-specific T cells
rotavius-specific B cells
rotavirus-specific SIg
1. Introduction
Vaccines against rotavirus (RV), until recently the main etiological agent of severe acute gastroenteritis (GE) in children, have been very successful (Yen et al., 2014). However, because of their lower efficacy in developing countries, where they are most needed, they still require improvement, and this will depend, in part, on our understanding of the adaptive immune response against the virus, a main interest of this research group (Angel et al., 2014). Immunity (Angel et al., 2012) and correlates of protection (Angel et al., 2014) to RV have been recently reviewed, and in the present chapter we will focus on our recent investigations regarding the immunity and correlates of protection to RV infection.
Multiple RV infections in the first few years of life result in virtually complete protection against moderate-to-severe gastroenteritis, but sterilizing immunity is not achieved (Angel et al., 2012). Recent data indicate that the number of RV infections (either symptomatic or asymptomatic) necessary to generate this protection are greater in children of low-income settings (Gladstone et al., 2011). These children also get their first infection earlier in life, which may contribute to the delayed protection because at least neutralizing antibodies responses are age dependent (Ward et al., 2006). Of importance for the understanding of RV immunity is the observation that RV induces both a transitory mucosal intestinal IgA response (likely related to protection) and persistent serological RV-specific IgM, IgG, and IgA responses (likely related to the antigenaemia and viraemia that acompany infection in children) (Angel et al., 2012). Moreover, although both serotype-specific and heterotypic immune responses are significant components of protective immunity against RV the latter probably plays a more important role (Angel et al., 2012).
Our studies of the adaptative immune response against RV in children and adults have shown that circulating human RV-specific memory lymphocytes have two unique characteristics: (1) both B (Jaimes et al., 2004; Rojas et al., 2007) and T (Rojas et al., 2003; Parra et al., 2014a) cells which are RV-specific preferentially express intestinal homing receptors. (2) Both B (Rojas et al., 2008; Narváez et al., 2012; Herrera et al., 2014) and T (Mesa et al., 2010; Parra et al., 2014b) RV-specific cells seem to have peculiar functional attributes: while the former are enriched in cells that have innate features, the latter have a poor functional profile. These characteristics are probably related to the fact that RV predominantly replicates in the intestinal microenvironment that is tolerogenic (Lamichhane et al., 2014), making this an excellent model to study lymphocytes from this compartment.
2. Rotavirus-specific T cells
2.1. Rotavirus-Specific CD4 T Cells
The understanding of RV-specific CD4 T-cell responses is a key issue because most of RV-specific antibody responses in the mouse model seem to be dependent on CD4 T-cells help (Franco and Greenberg, 1997). Although RV predominantly replicates in mature enterocytes of the small intestine, it also has a systemic dissemination and consequently, both intestinally and systemically primed T cells are expected to be elicited (Franco et al., 2006). Our main objectives for the study of human RV-specific T cells have been to characterize their phenotype and function, and to develop in vitro models to understand how they develop in an intestinal context.
Many functional experiments of RV-specific T cells included ELISPOT and intracellular cytokine staining assays (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002; Narváez et al., 2005). We found that healthy adults have circulating RV-specific CD4 T cells that secrete IFN-γ or IL-2, whereas cells producing IL-4, IL-13, IL-10, or IL-17 were below detection limits (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002; Narváez et al., 2005). The frequencies of RV-specific CD4 T cells producing IFN-γ in these subjects are comparable to those specific for other mucosal respiratory viruses (Mesa et al., 2010). However, the majority of RV-specific CD4 T cells were IFN-γ single producers, followed by a low percentage of double IFN-γ/IL-2 producers cells (Mesa et al., 2010), suggesting that T cells found in healthy adults are probably terminally differentiated effector cells, unable to provide long-term immunity. Analysis of the RV-specific CD4 T-cell stimulation with different RV antigens demonstrated that cellular responses of healthy adults were similar for cell culture adapted RV and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled in an IFN-γ ELISPOT (Kaufhold et al., 2005).
During acute and convalescence phases of RV infection intestinally primed RV-specific lymphocytes are expected to circulate in blood, on their way back to the intestine (see later). Our studies of RV-specific CD4 T cells circulating in children with acute gastroenteritis showed very low or below detection limit frequencies of circulating cells producing IFN-γ, compared to RV infection in healthy adults (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002). Additional cytokine producing RV-specific CD4 cells, such as IL-2+, IL-4+, IL-10+, IL-13+, or IL-17+, were below detection levels (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002). However, other research groups found increased levels of IL-6, IL-10, IFN-γ (Jiang et al., 2003), and TNF-α (Azim et al., 1999) in serum of acutely infected children. In blood of adults with RV gastroenteritis, virus specific CD4 T cells producing IFN-γ and low frequencies of those producing IL-10 and IL-2 were identified during acute and convalescence phases, respectively (Mesa et al., 2010; Jaimes et al., 2002).
In most of the studies from this laboratory, children with RV gastroenteritis were compared with children with gastroenteritis of other etiologies and only recently we typified circulating RV-specific CD4 T cells of a small number of healthy children (Parra et al., 2014b): most cells identified produced IFN-γ or TNF-α.
Multifunctional CD4 and CD8 T cells (proliferating and secreting various cytokines) are associated with protection against different pathogens (Mahnke et al., 2013; Seder et al., 2008). Since our results showed that the frequencies of RV-specific T cells producing IFN-γ are similar to those specific for mucosal respiratory viruses (Mesa et al., 2010), it was considered that the RV-specific T-cell responses may be poor mainly in terms of quality rather than quantity. To evaluate this hypothesis we recently compared proliferation and production of IFN-γ, TNF-α, and IL-2 by RV-specific T cells to cells specific for tetanus toxoid and influenza virus circulating in healthy adults and children (Parra et al., 2014b). Whereas tetanus toxoid- and influenza virus antigen-specific CD4 T cells are enriched in cells that produce two or more cytokines, RV-specific CD4 T cells are enriched in those producing only one cytokine (IFN-γ or TNF-α), (Parra et al., 2014b) (Fig. 2.9.1A). Besides, the frequencies of CD4 T cells producing cytokines and the proliferative responses were significantly higher after stimulation with tetanus toxoid and influenza virus antigens, compared to RV. Moreover, in the context of the linear functional differentiation model for Th1 CD4 T cells proposed by Seder et al. (Mahnke et al., 2013; Seder et al., 2008), RV-specific CD4 T cells are enriched in terminal effector memory populations, whereas those specific of tetanus toxoid and influenza virus antigens are more related with effector and central memory populations (Fig. 2.9.1B). Notably, the predominance of monofunctional RV-specific T cells was also detected in healthy children, signifying that this characteristic appears after infection at an early age, lasts until adult life, and may partially explain why immunity to RV is unable to provide long lasting protection (Parra et al., 2014b).
Figure 2.9.1 Comparison of the cytokine and differentiation profiles of antigen specific CD4 T cells.Fresh PBMC from healthy adults were cultured with tetanus toxoid, influenza virus antigen, and RV antigens in the presence of anti-CD28 and anti-CD49d monoclonal antibodies (MAbs) for 10 h; brefeldin A was added for the last 5 h of incubation. (A) After that, frequencies of live CD4 T cells producing one, two or three cytokines (IL-2, IFN-γ, and/or TNF-α) were evaluated by intracellular cytokine staining and flow cytometry (n = 13). (B) Frequencies of cytokine producing CD4 T cells in response to each antigen with a naïve (CCR7+ CD45RA+), central memory (CCR7+ CD45RA−), effector memory (CCR7− CD45RA−), and terminal effector memory (CCR7− CD45RA+) are represented (n = 3–5). (Source: Data were taken from publication Parra et al., 2014b.)
2.2. Rotavirus-Specific CD8 T Cells
In mice, RV-specific CD8 T cells provide the first mechanism, although not the only one, to clear a primary RV infection (Franco et al., 1997). Assessment of human circulating RV-specific CD8 T cells of healthy adults using ELISPOT and intracellular cytokine staining assays showed that the majority of these cells produced only IFN-γ, whereas cells producing both IFN-γ/IL-2 and those only producing IL-2 were rarely observed (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002; Narváez et al., 2005). In blood of adults with RV gastroenteritis virus-specific CD8 T cells producing IFN-γ were identified during acute and convalescence phases (Mesa et al., 2010; Jaimes et al., 2002). Compared to RV-infected and healthy adults, very low or below detection limit frequencies of circulating RV-specific CD8 T cells producing IFN-γ were identified in children with RV gastroenteritis (Rojas et al., 2003; Mesa et al., 2010; Jaimes et al., 2002), and they were below detection limit in healthy children (Parra et al., 2014b).
As for CD4 T cells, both the quality and magnitude of RV-specific CD8 T-cell responses in healthy adults were significantly lower than those specific for tetanus toxoid and influenza virus antigens (Parra et al., 2014b). In addition, RV-specific CD8 T cells also seem to be related to terminally differentiated memory cells (Mahnke et al., 2013) because the majority of cells produced only IFN-γ and had low proliferation capacity (Parra et al., 2014b).
2.3. Epitopes Recognized by Rotavirus-Specific T Cells
The fine specificity of the RV-specific T-cell response, either at a protein or epitope level, has been rarely investigated. In animal models, some epitopes have been identified in mice (Jaimes et al., 2005; Banos et al., 1997) and monkeys (Zhao et al., 2008).
Responses of T cells from healthy adults to a peptide pool based on the human VP6 sequence were compared with an IFN-γ ELISPOT (Kaufhold et al., 2005). In many cases the ELISPOT response to a human VP6 peptide pool was predictive of the subject’s overall response to individual RV strains. Thus, VP6 seems to be an important target of human RV-specific T cells. A RV-specific human T-cell epitope from VP7 protein (aa 40–52) restricted by HLA-DR4 (DRB1*0401) has been described, which stimulates proliferation of peripheral blood mononuclear cells (PBMC) from healthy adults and may be involved in molecular mimicry that could promote autoimmunity to pancreatic islet antigens (Honeyman et al., 2010).
We have recently identified three RV peptides restricted by HLA-DR1 (DRB1*0101) from viral proteins NSP2 (NSP2-3 SGNVIDFNLLDQRIIWQNWYA), VP3 (VP3-4 YNALIYYRYNYAFDLKRWIYL), and VP6 (VP6-7 DTIRLLFQLMRPPNMTPAVNA) that induce proliferation or production of IL-2, TNF-α, and/or IFN-γ by circulating CD4 T cells from healthy adults (Parra et al., 2014a). Of these, VP6-7 has all the characteristics of an epitope and totally overlaps with one previously found in mice (Banos et al., 1997) and partially with a VP6 epitope found in Rhesus macaques (Zhao et al., 2008), implying that this region is particularly prone to be recognized by CD4 T cells.
2.4. Markers of Intestinal Homing on Rotavirus-Specific T Cells
Since RV replication is highly restricted to enterocytes in vivo, the immune response against it originates in and exhibits its effector function directly at the intestinal mucosa. The homing of lymphocytes stimulated by antigens first encountered in intestinal Peyer’s patches back to the same or other Peyer’s patches or the intestinal lamina propria is mediated by interactions between the integrin α4β7 and CCR9 expressed on B and T lymphocytes and the cell adhesion molecule MadCAM 1 and CCL25, respectively, expressed on the vascular endothelium of the postcapillary venules in the intestine (Franco et al., 2006; Lee et al., 2014). Intestinal dendritic cells secreting retinoic acid imprint these receptors on T cells, which support trafficking properties based on the site- specific expression of their ligands and define a unique subset of CD4 memory T cells (Sigmundsdottir and Butcher, 2008). Initial studies of the expression of intestinal homing receptors by RV-specific T cells of healthy adults involved the proliferation and IFN-γ responses of circulating purified CD4 T cells expressing or lacking α4β7: Rott et al. (1997) found that purified α4β7+ CD45RA− cells proliferated more than 2.3-fold over α4β7− CD45RA− CD4 T cells to RV antigen. Subsequently we found that around 80% of RV-specific CD4 T cells producing IFN-γ expressed α4β7 (Rojas et al., 2003). Only recently, with the identification of DR1 restricted RV peptides and further production of HLA class II tetramers (Parra et al., 2014a), we were able to characterize the expression of both intestinal homing receptors, α4β7 and CCR9, on unpurified circulating RV peptide-specific CD4 T cells. As shown in Fig. 2.9.2, compared to influenza virus-specific tetramer+ CD4 T cells, RV-specific tetramer+ antigen experienced cells expressed both α4β7 and CCR9. Moreover, such cells were also detected in blood of two RV vaccinated children (Parra et al., 2014a). These results support the notion that small intestine originated cells are, indeed, a unique subset of CD4 T cells (Mahnke et al., 2013).
Figure 2.9.2 Intestinal migration markers on circulating influenza- and RV-specific CD4 T cells.RV- and influenza-specific CD4 T cells from eight healthy adults were identified using tetramers coupled with specific peptides and simultaneously stained with antibodies against α4β7 and CCR9. (Source: Data were taken from publication Parra et al., 2014a.)
2.5. Intestinal Human Rotavirus-Specific T Cells
Memory CD4 and CD8 T cells expressing intestinal homing receptor accumulate in the lamina propria, where they can reside for a long time. For this reason, it is possible that a bulk of RV-specific T cells remains in the intestine. However, studies with human intestines are difficult to perform because of the relatively high background CD4 and CD8 T cells present in an intracellular cytokine assay and only RV-specific CD8 T cells have been significantly detected (Narváez et al., 2010). Future studies with human intestinal RV-specific T cells will be important to establish whether they also have a poor functional profile similar to their circulating counterparts.
2.6. In Vitro Models to Study Human Rotavirus-Specific T Cells
We developed in vitro models to study the human systemic and intestinal RV immune response. The evaluation of systemic antigen presenting cells (APC) showed that monocyte derived dendritic cells (moDC) in contact with infectious RV are able to stimulate a strong CD4 Th1 allogeneic response (Narváez et al., 2005). Besides, circulating plasmacytoid dendritic cells are necessary to stimulate RV-specific memory T cells to produce IFN-γ (Mesa et al., 2007). Because moDC generally reflect the function of circulating peripheral blood myeloid dendritic cells, these findings predict that an important systemic Th1 response against RV should be generated under the viremic phase of infection. Since, as previously described, this is not the case, our results suggest that either the systemic antigen/virus that circulates during an acute infection with RV is presented to T cells in a tolerogenic context or that the amount of virus present systemically is unsuited for T-cell immunity induction.
The tolerogenic gut environment (Lamichhane et al., 2014) may substantially influence the T-cell response against RV. For the development of an in vitro model of human intestinal immune response against RV, we initially cultivated polarized Caco-2 cells in transwells and identified the “danger signals” released by cells infected by RV (Rodríguez et al., 2009). After infection, Caco-2 cells released IL-8, PGE2, small quantities of TGF-β1, and the constitutive and inducible heat shock proteins HSC70 and HSP70, which are known to induce a noninflammatory (non-Th-1) immune response (Rodríguez et al., 2009). Furthermore, HSC70, HSP70, and TGF-β1 were released, in part, associated with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation (Barreto et al., 2010). These MV were heterogeneous, with characteristics of exosomes and probably also of apoptotic bodies, and had immunomodulatory functions: MV from RV-infected cells induced death and inhibited proliferation of polyclonally stimulated CD4 T cells, and these effects were in part due to TGF-β (Barreto et al., 2010).
We next studied the effect of these intestinal immunomodulators in relation to the interaction of RV with moDC (Rodriguez et al., 2012): moDC treated with supernatants from RV-infected Caco-2 cells promoted a significantly lower Th1 response, in comparison with those treated with purified RV. Moreover, TGF-β, unlike thymic stromal lymphopoietin (TSLP), was an importat mediator of this modulation, suggesting that TGF-β could be an immune evasion mechanism (Rodriguez et al., 2012). In agreement with this hypothesis, in PBMC from healthy adults the inhibition of the TGF-β signaling pathway increased the frequency of RV-specific CD4 T cells that produce IFN-γ (Mesa et al., 2010). However, this inhibition was undetected in children, suggesting that RV-specific CD4 T cells could be modulated by other tolerogenic mechanisms, such as anergy.
We used three anergy inhibitors to assess the hypothesis of the presence of circulating anergic T cells in the response against RV: after stimulation of PBMC from healthy adults with RV in the presence of IL-2—unlike IL-12 or R59949 (a pharmacological diacyl- glycerol kinase alpha inhibitor)—increased frequencies of RV-specific CD4 and CD8 T cells producing cytokines were identified (Parra et al., 2014b). This finding depicts a poor functional T-cell profile that may be partially reversed in vitro by the addition of rIL-2.
The role of regulatory T cells (Treg) in RV infection has been investigated in few reports. In RV-infected mice, the numbers of FoxP3+ Treg cells are increased, but RV clearance or Abs levels are unsignificantly modified in their absence (Miller et al., 2014). In PBMC from healthy adults the depletion of CD25+ T cells (probably containing Treg cells) increases the frequency of RV-specific CD4 T cells that produce IFN-γ, suggesting that, at least systemically, Treg may modulate the function of RV-specific CD4 T cells (Mesa et al., 2010).
3. Rotavirus-specific B cells
A review of RV-specific B cells (Bc) has been recently published (Franco and Greenberg, 2013). Key to our studies of RV-specific Bc is a flow cytometry assay that detects specific binding of a fluorescent RV antigen to a B cell that expresses RV-specific Ig (Franco et al., 2006). In vitro, RV has been shown to induce activation and differentiation of human Bc, present in PBMC, into antibody secreting cells (ASC) (Narváez et al., 2010). However, this effect was undetected with purified Bc, suggesting the participation of other cells in activating the Bc; most likely dendritic cells that produce IFN-α (Deal et al., 2010).
Unexpectedly, a significant number (approximately 1–2%) of naïve IgD+ CD27– RV-specific Bc are detectable in cord blood (Parez et al., 2004). Although clearly specific, these cells secrete antibodies with a low affinity for VP6 (Kallewaard et al., 2008). Infants that have or lack serum RV-specific IgA (considered a hallmark of primary RV infection) have circulating RV-specific Bc that express IgM, IgD, and CD27, a phenotype compatible with memory B cells (mBc) (Rojas et al., 2007). In healthy adults, RV-specific mBc are enriched in all IgM Bc subsets, but in particular in the IgDlow IgMhi CD27+ subgroup of Bc (Narváez et al., 2012; Herrera et al., 2014) (Fig. 2.9.3). This phenotype is reminiscent of spleen marginal zone Bc, a subset that has been postulated to develop (by an unknown mechanism) a prediversified Ig repertoire and to participate in “innate” Ig responses to pathogens (Cerutti et al., 2013). Recently, in humans, it has been shown that circulating IgM mBc contains a population of cells that have characteristics of marginal zone Bc (Descatoire et al., 2014). Although the function of these cells remains unknown, in experiments in which human IgM mBc (mostly IgD + ) are passively transferred to RV infected immunodeficient mice, they are able to switch to IgG ASC and mediate immunity against RV antigenemia and viremia (Narváez et al., 2012).
Figure 2.9.3 RV-specific memory B cells (mBc) are enriched in the IgMhi IgDlow, IgMlow IgDhi, and IgM+ only mBc subsets.Summary of the median frequencies of seven subsets of total and RV-specific mBc assessed by multiparametric flow cytometry from 10 healthy adults. Donuts denote the median frequencies of CD19+ B cells (%) subsets. * Statistically significant differences (p < 0.05, two-tailed Wilcoxon test) between total and RV-specific mBc subsets. (Source: Data were taken from publication Herrera et al., 2014.)
As for memory T cells, both RV-specific ASC and mBc predominantly expressed α4β7 and CCR9. In children with an acute RV infection, the great majority (∼70%) of circulating virus specific Bc are ASC that coexpress both intestinal homing receptors (Jaimes et al., 2004). In the convalescent phase of viral infection, approximately one-third of RV-specific mBc express both homing receptors and are presumably targeted to the small intestine. Another third of RV-specific mBc only express α4β7 (presumably targeted to other parts of the intestine and other mucosal surfaces) and the final third express neither receptor (presumably targeted to the spleen and other systemic organs) (Jaimes et al., 2004; Gonzalez et al., 2003). Notably, like for RV-specific T cells described earlier, IgD− mBc that express these two markers are enriched compared to the total nonantigen specific lymphocytes [10 times for T cells (Parra et al., 2014a) and approximately 1.5 times for IgD− mBc (Jaimes et al., 2004)].
4. Correlates of protection
Correlates of protection for RV vaccines have been recently reviewed (Angel et al., 2014), and we focus here only on our most recent studies on this subject (Rojas et al., 2007; Herrera et al., 2013). Serum RV-specific IgA transiently reflect the intestinal RV-specific IgA and are, so far, the best practically measured correlate of protection against RV gastroenteritis in humans (Angel et al., 2012). Nevertheless, they are unsuitable to predict individual protection and therefore can be only used as an epidemiological tool at the population level (Cheuvart et al., 2014). The use of a validated correlate of protection as a surrogate endpoint after vaccination would contribute to the faster development of a new generation of RV vaccines and the assessment of its efficacy in a wider number of settings (Angel et al., 2014).
An adequate correlate of protection for RV could be a marker able to precisely reflect the intestinal immune response induction. Consistent with this is the finding that in children with an acute RV infection circulating IgD− RV-specific mBc express intestinal homing receptors (α4β7+, CCR9+) (Jaimes et al., 2004). In a double blind trial of the attenuated RIX4414 human RV vaccine (which contains the same vaccine strain virus present in the Rotarix formulation), we found correlations between protection from disease and frequencies of circulating RV-specific IgD− CD27+ α4β7+ CCR9+ mBc measured after dose 1 (D1) and levels of plasma RV-specific IgA after dose 2 (D2). However, other factors may be relevant for conferring protection from disease because correlation coefficients for both tests were low and protection against disease was significantly higher in vaccinees that lacked RV-specific IgA (titer < 1:100) compared to placebo recipients that also lacked these antibodies (Rojas et al., 2007).
Secretory Ig (SIg) in serum has been proposed as another method for indirectly measuring intestinal Ig (Grauballe et al., 1981). The polymeric immunoglobulin receptor on the basolateral membrane of the mucosal epithelial cells captures polymeric IgA and IgM (Mantis et al., 2011). This complex is endocytosed and transported to the apical membrane, where it is cleaved and part of it (the secretory component [SC]) remains attached to the Ig (IgA or IgM), forming SIg, which may be retro-transported across epithelial cells and ultimatelly enter the circulation (Mantis et al., 2011).
Given that RV-specific SIgs have been detected in serum of children with acute RV infection (Grauballe et al., 1981; Hjelt et al., 1985) and correlated with the amounts detected in duodenal fluid 1 week after the infection (Hjelt et al., 1986), we sought to confirm the presence of plasma RV-specific SIg in children with natural RV infection and to determine if circulating RV-specific SIg could more precisely reflect the intestinal protective immune response induced by the RIX4414 RV vaccine, and be a better correlate of protection than circulating RV-specific IgA after vaccination. Plasma samples collected from children with natural RV infection and from children who had received two doses of the RIX4414 RV vaccine or placebo were assessed by an in-house ELISA designed for meassuring plasma RV-specific SIg (Fig. 2.9.4). As shown in Figure 2.9.4, for this ELISA an antihuman SC monoclonal antibody is used as a capture antibody and thus, the assay does not discriminate between RV-specific SIgA and SIgM (Herrera et al., 2013).
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