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. 1998 Jan;72(1):191-200.
doi: 10.1128/JVI.72.1.191-200.1998.

DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice

Y L Lin  1 , L K Chen, C L Liao, C T Yeh, S H Ma, J L Chen, Y L Huang, S S Chen, H Y Chiang
Affiliations

DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice

Y L Lin et al. J Virol. 1998 Jan.

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1'), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1' could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1' coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.

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Figures

FIG. 1
FIG. 1
Schematic diagram of plasmid constructs expressing various JEV glycoproteins in the mammalian expression vector. The numbers with straight or bent arrows are the nucleotide positions on the JEV genome.
FIG. 2
FIG. 2
JEV glycoproteins expressed from recombinant plasmids in cell cultures. (A) JEV E protein expressed by pJME. Cell lysates from pJME-transfected (lane 2) or mock-transfected (lane 1) COS-7 were immunoblotted with MAb against JEV E protein as described in Materials and Methods. The position of the E protein is indicated by the arrow. Numbers on the left figure are the molecular mass standards in kilodaltons. (B) JEV NS1 proteins expressed by pJNS1 and pJNS1′ were immunoprecipitated with anti-NS1 MAb and analyzed by SDS-PAGE. Cell lysates were isolated from BHK-21 cells transfected with pJNS1 (lanes 2, 6, and 10), pJNS1′ (lanes 3, 7, and 11), or vector pcDNA3 (lanes 4, 8, and 12); as a control, lysates purified from JEV-infected BHK-21 cells (lanes 1, 5, and 9) were also included. The expression profiles of viral proteins analyzed by RIP are shown in lanes 1 to 4. For endo-F analysis, cell extracts either from JEV-infected cells or from transfected cells were digested with endo-F (lanes 5 to 8), or treated with buffer alone (lane 9 to 12) at 37°C overnight. Lane M contains the molecular weight standards (given in kilodaltons on the left). The positions of the glycosylated NS1 and NS1′ proteins are indicated by the arrows on the right. The asterisks denote the endo-F-sensitive species of NS1 glycoproteins.
FIG. 3
FIG. 3
Survival of DNA-immunized mice after lethal JEV challenge. Groups of 3- to 4-week-old female ICR mice were immunized with the indicated plasmids or buffer alone and later lethally challenged with JEV as described in Materials and Methods. The JEV-infected mice were monitored daily for survival up to 21 days postchallenge.
FIG. 4
FIG. 4
Seroconversion of mice immunized with pJNS1 or pJNS1′. The antibody responses specific to JEV NS1 in immunized mice were examined by RIP analysis with 35S-labeled, JEV-infected cell lysates. The pooled sera were collected 2 weeks after the primary (lanes 1 and 4), secondary (lanes 2 and 5), and third (lanes 3 and 6 to 8) DNA injections. The serum samples were from mice immunized with pJNS1 (lanes 1 to 3), pJNS1′ (lanes 4 to 6), vector control pcDNA3 (lane 7), or PBS buffer alone (lane 8). A MAb specific for JEV NS1 (αNS1) was used as a positive control (lane 9). The positions of NS1 and NS1′ are indicated by the arrows on the right. Numbers on the left are the molecular mass standards in kilodaltons.
FIG. 5
FIG. 5
The pJNS1-immunized sera exhibited antibody-dependent complement-mediated cytolysis of JEV-infected cells. The sera obtained 2 weeks after the second injections were pooled from ICR mice immunized with pcDNA3 (A), pJME (B), or pJNS1 (C). As a control, sera were also collected from mice that had been infected with JEV (D). In the presence of complement (C′) at different dilutions (none, 1:10, 1:20, or 1:40), the pooled sera were analyzed for their ability to lyse JEV-infected BHK-21 cells at various serum dilutions (no antibody, 1:20, 1:40, or 1:80). The percent specific lysis was determined as described in Materials and Methods.
FIG. 6
FIG. 6
Intracellular and extracellular protein patterns of JEV NS1 and NS1′ expressed in cell culture. (A) Viral proteins expressed from plasmids by a transient system involving recombinant vaccinia virus vTF7-3. By immunoblotting with MAb against JEV NS1, the culture media (lanes 1 to 4) and their corresponding cell lysates (lanes 5 to 8) were prepared to detect the presence of JEV NS1. Lanes: 1 and 5, samples derived from JEV-infected cells (positive control); 2 and 6, samples from pJNS1-transfected, vTF7-3-infected cells; 3 and 7, samples from pJNS1′-transfected, vTF7-3-infected cells; 4 and 8, samples from pcDNA3-transfected, vTF7-3-infected cells (negative control). Putative homo- and heterodimers of JEV NS1s are marked by arrows. (B) Viral proteins expressed by cell clones containing different plasmids as indicated. JEV NS1s in the culture media (lanes 1 to 4) and in cell lysates (lanes 5 to 8) were analyzed by immunoblotting. Samples to be examined were prepared from cell clones containing pJNS1 (lanes 2 and 6), pJNS1′ (lanes 3 and 7), and no plasmid (lane 4 and 8). Lanes 1 and 5 contain control samples derived from JEV-infected cells.
FIG. 7
FIG. 7
NS1 protein localization in NS1- or NS1′-expressing cell clones. By using indirect immunofluorescence staining of cells with MAb against JEV NS1, the NS1 expression patterns from cell clones containing pJNS1 (C and D) or pJNS1′ (E and F) were investigated. The NS1 pattern from JEV-infected BHK-21 cells (A and B) was included as a control. The intracellular distribution of NS1 was analyzed from cells fixed with acetone-methanol (1:1) (A, C, and E); on the other hand, cell surface expression of NS1 was analyzed from unfixed cells (B, D, and F) (see Materials and Methods).
FIG. 8
FIG. 8
Flow cytometry of NS1 surface expression from three permanent cell clones. NS1 antigen expressed on the surface of cell clones containing pJNS1 (A), pJNS1′ (B), or pJNS1-2A (C) was analyzed with a FACS Caliber (Becton Dickinson) and CELLQuest software. Cells were stained for surface antigen with a MAb against JEV NS1 followed by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (shaded areas). The open area was derived from the staining of parental BHK-21 cells. The numbers above the shaded areas in each panel indicate the percentage of positive staining from tested cells compared to that from parental BHK-21 cells.
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