Schematic diagram of the coagulation cascade and possible interference of different autoantibodies against dengue virus in the coagulatory pathway. The letters (A –L) in orange color represent autoantibodies against dengue virus proteins which may cross-react with coagulatory molecules through molecular mimicry as listed in Table 2. K, kallikrein; PK, prekallikrein; HK, high molecular weight kininogen; TFPI, tissue factor pathway inhibitor; APC, activated protein C; PAI-1, plasminogen activator inhibitor 1; FDP, fibrinogen deposition products. Dashed lines indicate inhibition 

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... between persistent symptoms and autoimmune-based disturbance exists. 30 This study showed that having more than one dengue infection could be a trigger for potential autoimmune phenomena. It is hypothesized that after dengue virus infection, cross- reacting antibodies to self-nuclear proteins are generated and a failure to properly clear the immune complexes may further cause an autoimmune post-dengue syndrome. 30 Hemorrhagic syndromes of DHF / DSS include thrombocytopenia, coagulopathy and vasculopathy, which are related to dysfunction of endothelial cells and platelets. 20,31 Dengue virus itself does not cause morphological damage to endothelium and vascular leakage is mediated indirectly by host factors induced by virus. 32 ADE-mediated infection of dengue virus in peripheral blood monocytes may modulate endothelial cell function via cytokines such as TNF- a . 33 Previous studies showed that dengue virus can infect endothelial cells, leading to chemokine production, complement activation and apoptosis. 34 In addition to dengue virus effects, it has been shown that mouse anti-dengue virus NS1 antibodies can bind to human endothelial cells. 35 Furthermore, autoantibodies present in dengue patient serum samples can cause endothelial cell damage. The endothelial cell binding activity and apoptosis induced by serum samples from DHF / DSS patients are higher than those induced by serum samples from DF patients. Antibodies against NS1 account, at least in part, for the cross-reactivity and apoptosis induction. 36 – 39 Furthermore, antibody-binding to liver endothelial cells and a hepatitis-like pathologic effect were also observed in mice after active immunization with NS1 or passive administration with anti-NS1 antibodies. 40 Anti-dengue antibody cross-reactivity to platelets has also been observed. 41,42 Serum samples from DHF / DSS patients show higher binding activity to platelets than do serum samples from DF patients. Furthermore, dengue patient serum samples cause complement-mediated platelet lysis and inhibit ADP-induced platelet aggregation. The findings of transient thrombocytopenia associated with the gener- ation of antiplatelet antibodies in dengue virus-infected mice 43 and the administration of anti-NS1 antibodies to mice 44 further support the hypothesis that cross-reactive antiplatelet antibodies may play a role in dengue pathogenesis. As previously proposed by Rose and Bona, 45 there are three types of evidence for autoimmunity in disease which include direct proof, indirect evidence and circumstantial evidence. The direct transfer of antibody that causes a disease is an example of direct proof. Maternally transferred dengue antibody is a known risk factor for infants who develop DHF on their first infection. This epiphenomenon has been used to justify the theory of ADE, but also may support the observed cross-reactive antibody binding to endothelial cells and platelets 37,41,42 as a cause of DHF. Although IgM in DHF patient serum samples plays a more dominant role than IgG in the cross-reactivity with platelets and endothelial cells, 37,41 antiplatelet IgG is present in DHF patient serum samples (unpublished obser- vations). It will be interesting to determine whether mater- nally transferred IgG antibody to infants is involved in DHF disease development. Furthermore, the categories of indirect evidence based on reproduction of the autoimmune disease in experimental animals 40,43,44 and circumstantial evidence from clinical clues 30 also support the hypothesis not only of ADE but also of autoimmunity as a contributory factor to DHF. Several autoantigens recognized by anti-NS1 antibodies have been identified by sequence alignment using NCBI and Swiss-Prot databases, including beta chain of H þ transporter / ATP synthase, protein disulfide isomerase (PDI), vimentin and heat shock protein 60. 46 Of note, anti-prM antibodies cross-reactive with BHK-21 or A549 epithelial cells also recognize heat shock protein 60. 47 Furthermore, anti-NS1-mediated platelet aggregation inhibition is, at least in part, attributable to its binding and inhibitory effect on PDI. 48 The C-terminal amino acid residues 311 – 352 of dengue virus NS1 shares sequence homology with host cell target proteins. Using a modified NS1 lacking cross-reactive epitopes, the C-terminal region of NS1 may be responsible for cross-reactivity with endothelial cells and platelets. 44,49 In addition, the deletion of the C-terminal region of dengue virus NS1 abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. 44 Since NS1 is a potential vaccine candidate for dengue, the NS1 epitopes that gener- ate cross-reactive antibodies will likely need to be removed or modified. Hemostasis is composed of four major events that occur following the loss of vascular integrity: (1) vascular constriction; (2) platelet activation and aggregation; (3) coagulation cascade and clot formation; and (4) clot dissol- ution. 50 As shown in Figure 1, the coagulation cascade con- sists of two pathways leading to fibrin formation: the intrinsic and extrinsic pathways. 51 The intrinsic pathway is initiated primarily by exposure of collagen to the blood vessel surface, while the extrinsic pathway is initiated by exposure of tissue factor upon vascular injury. The two pathways converge at the activation of factor X (FX) (gener- ating FXa, ‘a’ signifies active), and FXa forms a complex with factor Va (FVa) to activate prothrombin to thrombin. Thrombin converts fibrinogen to a fibrin network. Fibrinolysis is triggered by activation of plasminogen to plasmin by tissue plasminogen activator (tPA) or urokinase. Both procoagulatory and anticoagulatory molecules are tightly regulated to prevent unwanted thrombosis or bleeding under normal conditions. However, in some pathological conditions, such as antiphospholipid syndrome (APS), this delicate balance is broken by autoantibodies against coagulatory molecules. In APS patients, autoantibodies which cross-react with coagulatory molecules can be found in serum samples which may cause thrombosis and miscarriage. 52 – 55 It is still unknown by what mechanisms these cross-reactive autoantibodies are induced. However, molecular mimicry through bacterial or viral infection represents a popular and widely accepted hypothesis. 56,57 The presence of pathogen-induced autoantibodies cross- reactive with coagulatory molecules has been confirmed in two ways. One is direct sequence comparison of pathogen proteins and coagulatory molecules to identify a shared linear amino acid sequence. The other is conformational analysis to find a shared motif or domain structure. The homologous sequence or conformation between the host molecules and those of the pathogen must be different enough to be recognized as non-self by host immune cells. In addition, this shared region must be exposed so that antibodies against this region can bind to the target region to cause functional disturbance. 58,59 In the cases of coagulation and fibrinolysis-related molecules, because many of them such as coagulatory factor Xa, IXa, II (thrombin) and plasmin belong to the trypsin-like serine protease superfam- ily and share similar primary amino acid sequence and catalytic domain, 60 it is not surprising to find antibodies from APS patients that cross-react with many of these coagulatory molecules. 51 – 55 There are a group of viruses which can cause viral hemorrhage fever (VHF) during infections. 61,62 All these VHF viruses are RNA viruses with different transmission vectors and geographic distribution. However, they all show clinical manifestations of hemorrhage to various degrees. Among VHF, dengue, Marburg and Ebola are the most important ones, 62 and dengue virus is the most preva- lent in tropical and subtropical areas inhabited by its mos- quito vector. 63 Studies on the pathogenic mechanisms of dengue virus-induced hemorrhage may unveil some clues of the hemorrhagic conditions induced by the other VHF agents. In DHF / DSS patients, blood platelet numbers drop to less than 10 5 / mm 3 (thrombocytopenia) and there are signs of plasma leakage. 17 In addition, DHF patients have abnormal coagulopathy which is also evidenced by prolonged thrombin time (TT) and activated partial thromboplastin time, decreased fibrinogen levels and increased levels of fibrinogen degradation products. 17,64 Hemostatic changes in DHF / DSS involve multifactorial mechanisms which include not only vascular changes and thrombocytopenia but also coagulation disorders. 64 – 66 Several pieces of evidence suggest that autoantibodies may be involved in causing abnormal hemostasis during dengue virus infection. As mentioned above, antibodies against NS1 from both dengue patients and NS1-immunized mice can bind to endothelial cells and induce activation and apoptosis of endothelial cells. 36 – 39 In addition, anti-NS1 antibodies can also opsonize normal human platelets and enhance platelet- macrophage engagements in vitro . 67 Murine monoclonal antibodies against NS1 of dengue virus have also been shown to bind to human blood coagulation factors such as fibrinogen. 35 Moreover, like many coagulatory factors, dengue virus can also bind to heparin (although dengue is not unique among viruses in binding to heparin). 68 – 71 Taken together there is evidence to suggest that molecular mimicry between dengue virus and not only platelets and endothelial cells but also coagulatory molecules may also play important roles in the immunopathogenesis of dengue hemorrhage. A computer-based sequence comparison between dengue virus proteins and coagulatory molecules shows that at least 12 different regions of dengue virus proteins, including core, prM, E and NS1 proteins, have amino acid sequence simi- larity with different coagulatory molecules, such as factors X, XI, VII, etc. (Table 2). The possible interference of these antidengue antibodies in the coagulatory activation pathway is ...

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... 1, the coagulation cascade con- sists of two pathways leading to fibrin formation: the intrinsic and extrinsic pathways. 51 The intrinsic pathway is initiated primarily by exposure of collagen to the blood vessel surface, while the extrinsic pathway is initiated by exposure of tissue factor upon vascular injury. The two pathways converge at the activation of factor X (FX) (gener- ating FXa, ‘a’ signifies active), and FXa forms a complex with factor Va (FVa) to activate prothrombin to thrombin. Thrombin converts fibrinogen to a fibrin network. Fibrinolysis is triggered by activation of plasminogen to plasmin by tissue plasminogen activator (tPA) or urokinase. Both procoagulatory and anticoagulatory molecules are tightly regulated to prevent unwanted thrombosis or bleeding under normal conditions. However, in some pathological conditions, such as antiphospholipid syndrome (APS), this delicate balance is broken by autoantibodies against coagulatory molecules. In APS patients, autoantibodies which cross-react with coagulatory molecules can be found in serum samples which may cause thrombosis and miscarriage. 52 – 55 It is still unknown by what mechanisms these cross-reactive autoantibodies are induced. However, molecular mimicry through bacterial or viral infection represents a popular and widely accepted hypothesis. 56,57 The presence of pathogen-induced autoantibodies cross- reactive with coagulatory molecules has been confirmed in two ways. One is direct sequence comparison of pathogen proteins and coagulatory molecules to identify a shared linear amino acid sequence. The other is conformational analysis to find a shared motif or domain structure. The homologous sequence or conformation between the host molecules and those of the pathogen must be different enough to be recognized as non-self by host immune cells. In addition, this shared region must be exposed so that antibodies against this region can bind to the target region to cause functional disturbance. 58,59 In the cases of coagulation and fibrinolysis-related molecules, because many of them such as coagulatory factor Xa, IXa, II (thrombin) and plasmin belong to the trypsin-like serine protease superfam- ily and share similar primary amino acid sequence and catalytic domain, 60 it is not surprising to find antibodies from APS patients that cross-react with many of these coagulatory molecules. 51 – 55 There are a group of viruses which can cause viral hemorrhage fever (VHF) during infections. 61,62 All these VHF viruses are RNA viruses with different transmission vectors and geographic distribution. However, they all show clinical manifestations of hemorrhage to various degrees. Among VHF, dengue, Marburg and Ebola are the most important ones, 62 and dengue virus is the most preva- lent in tropical and subtropical areas inhabited by its mos- quito vector. 63 Studies on the pathogenic mechanisms of dengue virus-induced hemorrhage may unveil some clues of the hemorrhagic conditions induced by the other VHF agents. In DHF / DSS patients, blood platelet numbers drop to less than 10 5 / mm 3 (thrombocytopenia) and there are signs of plasma leakage. 17 In addition, DHF patients have abnormal coagulopathy which is also evidenced by prolonged thrombin time (TT) and activated partial thromboplastin time, decreased fibrinogen levels and increased levels of fibrinogen degradation products. 17,64 Hemostatic changes in DHF / DSS involve multifactorial mechanisms which include not only vascular changes and thrombocytopenia but also coagulation disorders. 64 – 66 Several pieces of evidence suggest that autoantibodies may be involved in causing abnormal hemostasis during dengue virus infection. As mentioned above, antibodies against NS1 from both dengue patients and NS1-immunized mice can bind to endothelial cells and induce activation and apoptosis of endothelial cells. 36 – 39 In addition, anti-NS1 antibodies can also opsonize normal human platelets and enhance platelet- macrophage engagements in vitro . 67 Murine monoclonal antibodies against NS1 of dengue virus have also been shown to bind to human blood coagulation factors such as fibrinogen. 35 Moreover, like many coagulatory factors, dengue virus can also bind to heparin (although dengue is not unique among viruses in binding to heparin). 68 – 71 Taken together there is evidence to suggest that molecular mimicry between dengue virus and not only platelets and endothelial cells but also coagulatory molecules may also play important roles in the immunopathogenesis of dengue hemorrhage. A computer-based sequence comparison between dengue virus proteins and coagulatory molecules shows that at least 12 different regions of dengue virus proteins, including core, prM, E and NS1 proteins, have amino acid sequence simi- larity with different coagulatory molecules, such as factors X, XI, VII, etc. (Table 2). The possible interference of these antidengue antibodies in the coagulatory activation pathway is shown in Figure 1. Many of these protein motifs are shared by different viruses of the Flaviviridae family which can cause different degrees of hemorrhage, such as Japanese encephalitis virus, Western Nile virus, yellow fever virus, tick-borne encephalitis virus and Omsk hemorrhagic fever virus (Figure 2). Hepatitis C virus which seldom causes hemorrhage, on the other hand, shows no similar motif at this region. Taken together, this region may play important roles in the pathogenesis of hemorrhage induced by these viruses. However, in yellow fever, the hemorrhage occurs during primary infection, while in DHF, most of the cases are secondary infection. Therefore, different mechanisms may be involved in the hemorrhage induced by flaviviruses, particularly with regard to the important role of antibody induced by dengue virus infection in DHF. Whether the protein sequences listed in Table 2 can induce antibodies during dengue virus infection is also largely unknown, although antibodies have been found in dengue patients which identify a sequence homology region between the E protein and plasminogen. There is also a correlation between plasminogen cross-reactive antibodies and hemorrhage in dengue patients. 74 In addition to sequence homology with plasminogen, amino acids 101– 106 of E protein (WGNGCG) shows sequence homology with factors XI, X, IX, VII, II (thrombin), plasminogen and tPA. In preliminary screening using serum samples from a previous study, 75 our unpublished data show that antibodies against factor Xa are also increased in DHF / DSS patients. Whether these antibodies which cross-react with coagulatory molecules contribute to the hemostatic abnormality and hemorrhage during DHF / DSS needs to be further studied. In addition to E protein, antibodies against NS1 have also been shown to cross-react with human fibrinogen. 35 However, the influence of these fibrinogen cross-reactive NS1 antibodies on fibrinogen activation remains unknown. Because serum samples generally contain many factors that can interfere with coagulation, it is necessary to separate the contributions of such factors from cross-reactive antibody effects. In this regard, the use of monoclonal antibodies may be of value. Different specificities of monoclonal antibodies may have different effects on coagulation molecules’ activation or function. An alternative way to study the influence of antibody on coagulation function is by using recom- binant single chain variable region (scFv), which retains the specificity of antibody but can be produced by bacterio- phage. We have generated scFv from NS1 immunized mice. Fibrinogen cross-reactive scFv antibodies were selected from the scFv library and tested for their effect on fibrinogen activation by TT. As shown in Figure 3, scFv against NS1 can interfere with fibrin formation which leads to prolonged TT. These data indicate that molecular mimicry between dengue virus and coagulatory molecules may be a result of cross- reactive autoantibodies which can interfere with coagulation activation. However, further studies are required to assess the importance of these autoantibodies in the development of hemorrhage during DHF / DSS. In summary, antibodies against dengue virus may interfere with hemostasis due to molecular mimicry between dengue virus proteins and coagulatory molecules. The epitopes recognized by these autoantibodies should be avoided in the design of candidate vaccines against dengue virus infection to avoid unwanted side-effects. Furthermore, the information gathered may provide clues to understand the mechanism of hemorrhage and develop better therapeutic strategies against VHF. As mentioned above, antibodies against dengue virus proteins such as NS1, prM or E can cross-react with platelets, endothelial cells and coagulatory molecules. 19,22,28 Molecular mimicry between dengue virus antigens and self- antigens may break self-tolerance. Plasma leakage in dengue patients is likely due to increased vascular permeability mediated by endothelial cells. Endothelial cell perturbation occurs by a variety of mechanisms, such as cytokine-mediated effects, direct virus cytopathy and anti- endothelial cell antibody-mediated pathology. In addition, platelets are destroyed or functionally impaired by cross- reactive antibodies. Macrophages are activated by dengue virus or cytokines such as IFN- g during the acute phase of infection. Activated macrophages may then phagocytose autoantibody-opsonized platelets and endothelial cells, and thus contribute to the development of thrombocytopenia and plasma leakage in DHF / DSS. 19,28,76 Antidengue cross-reactive antibodies to platelets and endothelial cells provide an explanation for the target specificity and unique feature of thrombocytopenia and plasma leakage during the development of DHF / DSS. In addition, antibodies against dengue virus may interfere with hemostasis due to the molecular mimicry between dengue virus proteins and coagulatory ...