Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fc γ Receptor-Dependent and -Independent Mechanisms

Abstract

The flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc ␥ receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q-and Fc ␥ receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.West Nile virus (WNV) is a single-stranded, positive-senseenveloped RNA virus that is maintained in nature through a mosquito-bird-mosquito transmission cycle. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks now occur annually in North America. Humans, which are dead-end hosts, can develop a febrile illness that progresses to a meningitis or encephalitis syndrome (32). At present, treatment is supportive, and no vaccine exists for human use.A member of the Flaviviridae family, WNV is closely related to other major human pathogens such as yellow fever (YF), dengue (DEN), tick-borne encephalitis (TBE), Japanese encephalitis (JEV), and Murray Valley encephalitis (MVE) viruses. The 10.7-kilobase genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by both virus-and host-encoded proteases (5). The NS proteins include an RNA-dependent RNA polymerase (NS5), a helicase/protease (NS3), and other proteins that form part of the viral replication complex (36, 37).NS1 is a highly conserved 48-kDa glycoprotein with 12 invariant cysteine residues. Although the disulfide linkage arrangement of MVE and DEN NS1 has been described (4, 66), structural analysis is currently lacking. NS1 is inserted into the lumen of the endoplasmic reticulum via a signal peptide that is cleaved cotranslationally by a cellular signalase to gene...

Preparation and immunogenic properties of a recombinant West Nile subunit vaccine

“…The inclusion of NS1 in the vaccine may provide further benefit by generating additional antibodies and/or T cells capable of protective activities. A recent report has documented both complement and Fcγ receptordependent and -independent protective activities of antibodies to WNV NS1 [41]. In our ongoing studies on a recombinant subunit vaccine for dengue virus, the addition of NS1 to envelope protein in the vaccine increased the production of IFN-γ from antigen-stimulated immune mouse splenocytes in vitro (Lieberman, MM et al, unpublished data).…”

“…This cellular immune response may contribute significantly to the overall protective efficacy of a subunit vaccine. In addition, there is evidence that NS1 may elicit a humoral protective immune response involving the complement fixing activity of antibodies to this protein [29,30], through mechanisms, such as antibody-dependent, complement-mediated cytolysis, or Fc receptor mediated antibody-dependent cellular cytotoxicity [30]. Thus, the inclusion of NS1 in the vaccine formulation can be justified on the basis of a humoral as well as a cellular immune response.…”

Anti-inflammatory activity of intravenous immunoglobulins protects against West Nile virus encephalitis

“…IVIG has a broad repertoire of neutralizing antibodies for various pathogens and neutralization is commonly assumed to be the mechanism of protection against viral infection. However, we have shown in the HSV1 model that IVIG devoid of neutralizing antibodies is as effective as IVIG in protection against encephalitis (Ramakrishna et al, 2011) and the reports that non-neutralizing antibodies can protect against WNV infection are consistent with this (Chung et al, 2006;Mehlhop et al, 2005;Vogt et al, 2011). Studies investigating IVIG protection from WNV infection have based dosing on its neutralizing activity and have thus used IVIG at suboptimal doses (0.1-0.6 g kg 21 ) to elicit antiinflammatory responses.…”

Flavivirus nonstructural protein NS1: Complementary surprises

“…A key question that arises from their findings is how they might relate to NS1 as the target of protective antibodies. Earlier work from the same laboratory linked the activity of some strongly protective anti-WNV NS1 monoclonal antibodies to host Fc␥ receptor (Fc␥R) function, although at least one antibody conferred solid protection in Fc␥R and C1q (classical complement pathway)-deficient mice (26). In light of Chung et al's (9) current findings, it seems reasonable to speculate that protective anti-NS1 antibodies might also operate by blocking fH attachment to NS1.…”

“…The novel finding of Chung et al (9) originated in the course of preparing WNV recombinant NS1 for monoclonal antibody development (26). A Ϸ150-kDa protein was copurified with rNS1 if FBS was present in the system.…”