Identification of three novel CTL epitopes within nucleocapsid protein of Hantaan virus.

Hantaan virus (HTNV) is a member of the Hantavirus genus that causes human hemorrhagic fever with renal syndrome (HFRS) in humans. The CTL response seems to play a key role in control of viral infection, but only a few HTNV epitopes recognized by the CTLs have been reported. Herein, we screened a panel of overlapping peptides covering the HTNV nucleocapsid protein by ELISPOT assays for those that can elicit IFN-γ production in vitro. Three novel CD8(+) CTL epitopes, N197-205 (RYRTAVCGL), N245-253 (KLLPDTAAV), and N258-266 (GPATNRDYL), were defined on the nucleocapsid protein and were found to be restricted by various HLA alleles including A11, A24, and B7. The epitopes were highly conserved among the reported HTNV strains and other hantanviruses, including Dobrava-Belgrade virus and Seoul virus, supporting their potential use in vaccine designs.

Fusion with extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 leads to enhancement of immunogenicity of Hantaan virus DNA vaccines in C57BL/6 mice.

BACKGROUND: Hantaan virus (HTNV) is the causative agent of the most severe form of a rodent-borne disease known as hemorrhagic fever with renal syndrome (HFRS). A safe and effective HTNV vaccine is needed. Vaccination with DNA constructs expressing fused antigen with bioactive factors, has shown promising improvement of immunogenicity for viral agents in animal models, but the effect of fusion strategy on HTNV DNA vaccine has not been investigated. RESULTS: DNA plasmids encoding the HTNV nucleocapsid protein (N) and glycoprotein (Gn and Gc) in fusion to the extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 (eCTLA-4) targeting to antigen presenting cells (APCs) were constructed. Intramuscular immunization of mice with plasmids expressing eCTLA-4-HTNV-N/GP fusion proteins leads to a significant enhancement of the specific antibody response as well as cytotoxic T-lymphocyte (CTL) response in C57BL/6 mice. Moreover, this effect could be further augmented when co-administered with CpG motifs. CONCLUSIONS: Modification of viral antigen in fusion to bioactive factor will be promising to confer efficient antigen presentation and improve the potency of DNA vaccine in mice.

Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5' termini of their genomic RNAs are monophosphorylated.

dsRNA and 5'-triphosphate RNA are considered critical activators of the innate immune response because of their interaction with pattern recognition receptors. It has been reported that no dsRNA is detected in negative-sense RNA virus-infected cells and that Hantaan virus (HTNV) genomic RNA bears a 5' monophosphate group. In this paper we examine the 5' termini of genomic RNAs of and dsRNA production by two major groups of Old World hantaviruses. No detectable amounts of dsRNA were found in infected cells. Also, the genomic RNAs of these hantaviruses bear a 5' monophosphate group and therefore are unable to trigger interferon induction. Taken together with the earlier data on HTNV, these results suggest that in addition to the dsRNA and genomic RNA, which may be only minimally involved in the induction of innate immunity, other cellular signalling pathways may also be involved and that these await further investigation.

Identification of oligopeptides mimicking the receptor-binding domain of Hantaan virus envelope glycoprotein from a phage-displayed peptide library.

Attachment of enveloped viruses to cells is triggered by the receptor-binding domain (RBD) on envelope glycoproteins (GP) binding to receptors located on the cell surface. To date, recognized receptors and RBD of hantaan virus (HTNV) have not been exactly defined. In this study, one monoclonal antibody (MAb) 3G1 possessing high neutralizing activity, which is directed against HTNV envelope glycoprotein G2, was used to determine the crucial motif of RBD. Peptide ligands binding to MAb 3G1 were selected from a 12 amino acid peptide library displayed on filamentous phages. After 3 rounds of selection, the binding capacity between phages and MAb 3G1 was examined byELISA. Afterwards the positive phage clones with high binding activity to MAb 3G1 were chosen and sequenced. The peptide sequences of positive phage clones were compared with that of HTNV 76-118 strain G2. A motif Y/F/WPW(X)HX1-2HY, aligned to the primary sequences of G2 96YPWHTAKCHY105, was identified from the peptide inserts in the 9 positive clones. Positive phages and synthesized peptide containing the motif were bound significantly to virus-susceptible cell (Vero-E6) membranes by ELISA and immunofluorescence assay, respectively. Therefore, the sequence on G2 between amino acid 96 and 105 may be a key motif of HTNV RBD recognized by viral receptors on target cell membranes. Further characterization of the motif would provide useful information in understanding of the cellular entry of HTNV.

Analysis of the immune response to Hantaan virus nucleocapsid protein C-terminal-specific CD8(+) T cells in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

[Identification of a functional ITAM-like sequence within G1 cytoplasmic tail of Hantaan virus].

The G1 cytoplasmic tail of Hantaan virus (HTNV) harbors a highly conserved region, which is homologous to immunoreceptor tyrosine-based activation motifs (ITAM) and is termed the ITAM-like sequence. To demonstrate the potential signal-transducing activity of G1 ITAM-like sequence resembling the canonical ITAM within immune and endothelial cells, a series of experiments were performed to define its interaction with cellular kinases. The synthesized G1 ITAM-like peptide was shown to coprecipitate with cellular phosphoprotein complexes by an immune-complex kinase assay. Mutational analyses showed that this ITAM-like sequence was a substrate for the Src family kinase Fyn, and two conserved tyrosine residues were required for coprecipitating Lyn, Syk, and ZAP-70 kinases. These findings demonstrated that HTNV envelope glycoprotein G1 contains a functional ITAM-like sequence in its cytoplasmic tail, which can bind critical cellular kinases that regulate immune and endothelial cell functions.

Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion.

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.

Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking.

The membrane glycoproteins Gn and Gc of Hantaan virus (HTNV) (family Bunyaviridae) are modified by N-linked glycosylation. The glycoproteins contain six potential sites for the attachment of N-linked oligosaccharides, five sites on Gn and one on Gc. The properties of the N-linked oligosaccharide chains were analyzed by treatment with endoglycosidase H, peptide:N-glycosidase F, tunicamycin, and deoxynojirimycin and were confirmed to be completely of the high-mannose type. Ten glycoprotein gene mutants were constructed by site-directed mutagenesis, including six single N glycosylation site mutants and four double-site mutants. We determined that four sites (N134, -235, -347, and -399) on Gn and the only site (N928) on Gc in their ectodomains are utilized, whereas the fifth site on Gn (N609), which faces the cytoplasm, is not glycosylated. The importance of individual N-oligosaccharide chains varied with respect to folding and intracellular transport. The oligosaccharide chain on residue N134 was found to be crucial for protein folding, whereas single mutations at the other glycosylation sites were better tolerated. Mutation at glycosylation sites N235 and N399 together resulted in Gn misfolding. The endoplasmic reticulum chaperones calnexin and calreticulin were found to be involved in HTNV glycoprotein folding. Our data demonstrate that N-linked glycosylation of HTNV glycoproteins plays important and differential roles in protein folding and intracellular trafficking.

[Construction and stable expression of intracellular antibodies to glycoprotein of hantavirus].

OBJECTIVE: To understand the molecular mechanisms of hantavirus assembly and maturation by stably expressing the intracellular antibodies to hantavirus glycoprotein G1 and G2 in endoplasmic reticulum (ER) and cytoplasm (Cyto) of Vero E6 cell. METHODS: The genes of VH and VL of antibodies against glycoprotein of hantavirus were amplified by PCR and cloned into pOPE 101-215 (Yol) vector. The G1 and G2 proteins specific ScFv genes were first expressed in E.coli and the function and binding properties were identified. The gene of ScFv were further inserted into intracellular expression vectyrs pEF/ myc/ ER and pEF/ myc/ CYTO vector and transfected Vero E6 cell. The clonal cell line which stabl expresses ScFv were isolated under the pressure of G418. RESULTS: The ScFv genes of hantavirus G1 and G2 specific antibodies were successfully expressed in subcellular compartment ER and Cyto of Vero E6 cells and specifically targeted G1 and G2 protein after virus infection of the cells. CONCLUSIONS: The recombination of intrabody to Hantann virus glycoprotein was constructed successfully, and it may provide basic material for the studying antiviral gene therapy and the molecular mechanism of viral replication and infection.

Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells.

We have developed a cell culture-derived, inactivated vaccine against Hantaan virus for prevention of the hemorrhagic fever with renal syndrome (HFRS). Hantaan virus was purified from a microcarrier culture of Vero E6 cells by ultrafiltration and density gradient centrifugation. Viral infection was inactivated by treatment of the viral stock with formaldehyde. Immunogenic properties of the vaccine were characterized in comparison with Hantavax, a mouse brain-derived, formalin-inactivated vaccine that has been in human use for a decade in Korea. Compared to the Hantavax, immunization of Balb/c mice with the cell culture-based vaccine resulted in a moderate difference in antibody response to the viral nucleocapsid protein but more than five-fold increase in neutralizing activity. Moreover, all six mice immunized with 5 microg of the cell culture-based vaccine were fully protected from challenge with infectious virus, whereas virus was detected in lung and spleen of all animals immunized with the same dose of Hantavax. Four times higher dose of the latter vaccine was needed for complete protection. In the analysis of the humoral immune response to the vaccines, we found that all three viral structural proteins, N, G1 and G2 were immunoprecipitated by sera from animals immunized with the cell culture-based vaccine. In contrast, N and some G1 but no G2 were precipitated by the sera from animals immunized with Hantavax. These results suggest that the cell culture-based vaccine can provide more effective immunity than the Hantavax.

Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test.

A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4 degrees C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA.

The medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted glycoprotein precursor GPC. We have demonstrated here the expression of the glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif "WAASA." Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus glycoproteins.

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

Nucleotide sequence of nucleocapsid protein (N) of Hantaan virus isolated from a Korean hemorrhagic fever patient.

The nucleotide sequence of the nucleocapsid protein (N) coding region of a Hantaan virus strain (CFC94-2) isolated from a Korean Hemorrhagic Fever (KHF) patient was determined by sequencing a series of deletion mutants. Comparison of the N coding sequence of CFC94-2 to the sequence of the prototype of Hantaan virus, strain 76-118 reveals a 4.97% difference in the nucleotide sequence and a 1.6% difference in the deduced amino acid sequence. The rate of amino acid sequence variation in N protein of different Hantaan viruses (1.6%) is quite similar to that in G1 and G2 envelope proteins (1.7%). These results suggest that N protein may be under a similar selection pressure to G1 and G2 envelope proteins against host immune system.

Development of monoclonal antibodies against Hantaan virus nucleocapsid protein.

Forty-five hybridoma cell lines producing monoclonal antibodies against Hantaan virus, the etiologic agent of hemorrhagic fever with renal syndrome, were generated by fusion of P3-X63-Ag8.V653 myeloma cells with spleen cells of mice immunized with inactivated Hantaan virus vaccine. Among these, 38 antibodies were identified as binding to the 48-kDa nucleocapsid protein by immunoblot assay or radioimmunoprecipitation. Twenty-six of them were of the immunoglobulin G1 (IgG1), nine were of the IgG2a, and three were of the IgA isotype. According to cross-reactivities with other serotypes of the genus Hantavirus, the antibodies were classified into three groups: 6 antibodies specific to the Hantaan serotype (group I), 20 antibodies cross-reacting with Hantaan and Seoul serotypes (SR-11, Tchoupitoulas, and R22) (group II), and 12 antibodies cross-reacting with Hantaan, Seoul, and Prospect Hill serotypes (group III). None of the antibodies cross-reacted with the Puumala serotype. With a panel of antibodies of different cross-reactivities, serotypes of Hantavirus could be differentiated. Thirty-eight monoclonal antibodies against Hantaan virus nucleocapsid protein which have different cross-reactivities between serotypes were developed. These results confirmed the presence of multiple serotype-specific epitopes on the nucleocapsid protein of Hantaan virus, which can be utilized in differentiation of serotypes.