Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.The genus Flavivirus, many of whose more than 70 members are arthropod-borne human pathogens, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus, and Japanese encephalitis virus (JEV), has assumed increasing public health importance in recent years. The single-strand, positive-sense RNA genomes of flaviviruses encode a single polyprotein, which is cotranslationally cleaved to produce three structural proteins (capsid C], membrane M], and envelope E]) and seven nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). NS1, a glycoprotein, is perhaps the most versatile among these, being involved both in vital processes such as viral RNA synthesis and in multiple interactions with the host, in ways that appear to benefit both pathogen and host. Following translocation into the lumen of the endoplasmic reticulum through a hydrophobic signal sequence that is encoded by the carboxyl terminus of E (17), NS1 undergoes glycosylation followed by rapid dimerization (44, 52). In DENV infection of cultured mammalian cells, extracellular NS1 was additionally detected as hexamers (19, 51). Despite the apparent absence of a canonical hydrophobic membrane anchor domain, the NS1s from JEV, Kunjin virus, DENV, and YFV have all been shown to be present on the surface of virus-infected cells (8, 23, 50). The mechanistic details of this membrane anchor still remain uncertain.The ability of DENV NS1 to bind host complement (9, 49) pointed to a role for this protein in DENV pathogenesis. Serum NS1 levels in both DENV and WNV patients correlate directly with disease severity (1, 36). Promotion of immune complex formation (54), ability to elicit autoantibodies with reactivity to platelets and extracellular matrix (10), and damage inflicted on endothelial cells (34) are some of the mechanisms proposed to explain pathogenesis mediated by DENV NS1. Recent studies with WNV NS1 demonstrated its ability to bind human complement factor H, suggesting a role in reducing the host''s ability to bring about complement-mediated control of early virus replication (11).Critical differences between the functions of NS1s encoded by different pathogenic flaviviruses and their contributions to pathology are evident from the published reports, with DENV NS1 believed to be involved in complement activation and the consequent capillary leak syndrome of dengue hemorrhagic fever (6), while WNV NS1 appears relatively more benign and has more to do with modulation of the host innate immune response (11). We have not encountered reports of adverse impacts of JEV NS1 in infected individuals.Paradoxically, several studies have pointed to a role for flavivirus NS1-specific immune responses in protection against flaviviruses. Passive immunization studies using monoclonal antibodies (MAbs) (24, 28, 29, 55) as well as immunization of mice using naked DNA constructs expressing NS1 (35, 40) revealed that antibodies directed to prM or E of DENV and NS1 of DENV and JEV are protective. Studies by different groups have shown that active immunization with purified NS1 or passive immunization with MAbs against YFV and DENV NS1 provides protection from lethal viral challenge in the absence of neutralizing antibodies (24, 45, 48). A panel of anti-WNV NS1 MAbs revealed multiple antibody-mediated mechanisms for protection, some mediated through complement and others via the Fc receptor (12). Those authors went on to show that anti-NS1 MAbs that facilitate phagocytosis and clearance of WNV-infected cells through Fc-γ receptors I and/or IV belonged to the IgG2a subclass and bound to cell surface-associated NS1 (13).Earlier studies also pointed to the cytolytic potential of NS1 antibodies, a property that might contribute significantly to their protective ability. Passive immunization experiments using a panel of anti YFV NS1-specific MAbs showed a significant correlation between protection and in vitro complement-mediated cytolysis of YFV-infected mouse neuroblastoma cells (47). Additionally, immunization of mice with a DNA vaccine construct carrying JEV NS1 induced a strong antibody response exhibiting complement-mediated cytolysis of JEV-infected cells (35), but no neutralizing activity, and resulted in protection against subsequent challenge with virus. Cell-mediated immune responses directed to NS1 of JEV have also been reported to play a role in cytotoxic T-lymphocyte-mediated killing of JEV-infected murine target cells (41). Thus, NS1 appears to contribute to protection in the murine model by inducing both humoral and cell-mediated arms of the immune response.It was therefore of interest to query whether NS1-specific antibodies in humans exposed to JEV exhibit cytolytic activity and to determine if these antibodies are capable of reducing virus production by infected cells. In this study we report for the first time the existence of detectable levels of anti-NS1 antibodies in a significant proportion of sera from humans infected with JEV and demonstrate their ability to induce antibody-dependent complement-mediated cytolysis of cells expressing JEV NS1 on the surface. These sera failed to cause lysis of cells infected with WNV or DENV, both of which cocirculate with JEV in the Indian subcontinent and have been reported in the region where we enrolled our volunteers, revealing stringent specificity and absence of flaviviral cross-reactivity for these cytolytic antibodies. Furthermore, we demonstrate the ability of NS1-specific antibodies elicited in mice to limit virus production in infected human SW-13 cell monolayers, which may explain, at least in part, the widely reported protective ability of flavivirus NS1. Significantly, we found no evidence for the ability of NS1 from JEV to bind human complement factor H, in contrast to the case for WNV NS1 (11). Taken together, these findings suggest that JEV NS1 may positively and significantly affect virus-specific protective immune responses.