Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies

We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.     

West Nile virus envelope protein inhibits dsRNA-induced innate immune responses

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.     

Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein

Using a panel of neutralizing monoclonal antibodies, we have mapped epitopes in domain III of the envelope protein of the New York strain of West Nile virus. The ability of monoclonal antibodies that recognize these epitopes to neutralize virus appeared to differ between lineage I and II West Nile virus strains, and epitopes were located on the upper surface of domain III at residues E307, E330, and E332.     

Identification of immunodominant epitopes derived from the respiratory syncytial virus fusion protein that are recognized by human CD4 T cells

Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.     

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.     

West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.     

Antecedent avian immunity limits tangential transmission of West Nile virus to humans

Background

West Nile virus (WNV) is a mosquito-borne flavivirus maintained and amplified among birds and tangentially transmitted to humans and horses which may develop terminal neuroinvasive disease. Outbreaks typically have a three-year pattern of silent introduction, rapid amplification and subsidence, followed by intermittent recrudescence. Our hypothesis that amplification to outbreak levels is contingent upon antecedent seroprevalence within maintenance host populations was tested by tracking WNV transmission in Los Angeles, California from 2003 through 2011.

Methods

Prevalence of antibodies against WNV was monitored weekly in House Finches and House Sparrows. Tangential or spillover transmission was measured by seroconversions in sentinel chickens and by the number of West Nile neuroinvasive disease (WNND) cases reported to the Los Angeles County Department of Public Health.

Results

Elevated seroprevalence in these avian populations was associated with the subsidence of outbreaks and in the antecedent dampening of amplification during succeeding years. Dilution of seroprevalence by recruitment resulted in the progressive loss of herd immunity following the 2004 outbreak, leading to recrudescence during 2008 and 2011. WNV appeared to be a significant cause of death in these avian species, because the survivorship of antibody positive birds significantly exceeded that of antibody negative birds. Cross-correlation analysis showed that seroprevalence was negatively correlated prior to the onset of human cases and then positively correlated, peaking at 4–6 weeks after the onset of tangential transmission. Antecedent seroprevalence during winter (Jan – Mar) was negatively correlated with the number of WNND cases during the succeeding summer (Jul–Sep).

Conclusions

Herd immunity levels within after hatching year avian maintenance host populations <10% during the antecedent late winter and spring period were followed on three occasions by outbreaks of WNND cases during the succeeding summer. Because mosquitoes feed almost exclusively on these avian species, amplification was directly related to the availability of receptive non-immune hosts.     

A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.     

A combination of naturally occurring mutations in North American West Nile virus nonstructural protein genes and in the 3' untranslated region alters virus phenotype

We previously reported mutations in North American West Nile viruses (WNVs) with a small-plaque (sp), temperature-sensitive (ts), and/or mouse-attenuated (att) phenotype. Using an infectious clone, site-directed mutations and 3' untranslated region (3'UTR) exchanges were introduced into the WNV NY99 genome. Characterization of mutants demonstrated that a combination of mutations involving the NS4B protein (E249G) together with either a mutation in the NS5 protein (A804V) or three mutations in the 3'UTR (A10596G, C10774U, A10799G) produced sp, ts, and/or att variants. These results suggested that the discovery of North American WNV-phenotypic variants is rare because of the apparent requirement of concurrent polygenic mutations.     

Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.     

Glycosylation of immunodominant linear epitopes in the carboxy-terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses

This study evaluated type-specific and cross-reactive neutralizing antibodies induced by immunization with modified surface glycoproteins (SU) of the 63 isolate of caprine arthritis-encephalitis lentivirus (CAEV-63). Epitope mapping of sera from CAEV-infected goats localized immunodominant linear epitopes in the carboxy terminus of SU. Two modified SU (SU-M and SU-T) and wild-type CAEV-63 SU (SU-W) were produced in vaccinia virus and utilized to evaluate the effects of glycosylation or the deletion of immunodominant linear epitopes on neutralizing antibody responses induced by immunization. SU-M contained two N-linked glycosylation sites inserted into the target epitopes by R539S and E542N mutations. SU-T was truncated at 518A, upstream from the target epitopes, by introduction of termination codons at 519Y and 521Y. Six yearling Saanen goats were immunized subcutaneously with 30 microg of SU-W, SU-M, or SU-T in Quil A adjuvant and boosted at 3, 7, and 16 weeks. SU antibody titers determined by indirect enzyme-linked immunosorbent assay demonstrated anamnestic responses after each boost. Wild-type and modified SU-induced type-specific CAEV-63 neutralizing antibodies and cross-reactive neutralizing antibodies against CAEV-Co, a virus isolate closely related to CAEV-63, and CAEV-1g5, an isolate geographically distinct from CAEV-63, were determined. Immunization with SU-T resulted in altered recognition of SU linear epitopes and a 2.8- to 4.6-fold decrease in neutralizing antibody titers against CAEV-63, CAEV-Co, and CAEV-1g5 compared to titers of SU-W-immunized goats. In contrast, immunization with SU-M resulted in reduced recognition of glycosylated epitopes and a 2.4- to 2.7-fold increase in neutralizing antibody titers compared to titers of SU-W-immunized goats. Thus, the glycosylation of linear immunodominant nonneutralization epitopes, but not epitope deletion, is an effective strategy to enhance neutralizing antibody responses by immunization.     

Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.     

Modification of nonstructural protein 1 of influenza A virus by SUMO1

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.     

The reovirus nonstructural protein sigma1NS is recognized by murine cytotoxic T lymphocytes.

The cytotoxic T-lymphocyte (CTL) response in reovirus-infected C3H mice was investigated by using reovirus-vaccinia virus recombinants. Results of cytotoxicity assays indicated that the nonstructural protein sigma1NS elicited a significant CTL response. Experiments with sigma1NS-specific CTL lines showed that both strain-specific and cross-reactive epitopes exist in the sigma1NS protein.     

Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms

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 gamma receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q- and Fc gamma receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.     

Immunochemical properties of West Nile virus protein prM and protein M C-end

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.