Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

The 293T cell line is derived from human embryonic kidney cell line 293 and contains the simian virus 40 large T antigen. 293T cells were maintained in Dulbecco modified Eagle medium (Nissui, Tokyo, Japan) supplemented with 0.45% glucose and 10% heat-inactivated fetal calf serum (FCS). The E6 clone of Vero cells (ATCC C1008; Cell Repository line 1586) was grown in Eagle minimal essential medium (Nissui) supplemented with 2 mM glutamine and 5% heat-inactivated FCS.

cDNA clones containing the coding region of the M segment of HTNV strain 76-118 and SEOV strain SR-11 were kindly provided by C. S. Schmaljohn. The cDNA of the M segment of HTNV was excised by using BglII and subcloned into the BglII site of expression vector pCAGGS/MCS (15). The cDNA of the M segment of SEOV was excised by using SmaI and XhoI and subcloned into the SmaI and XhoI sites of pCAGGS/MCS. The plasmids containing the M segment cDNA of HTNV and SEOV were designated pCHTNM and pCSEOM, respectively. pCVSVG is the expression plasmid made by cloning the cDNA encoding the VSV G protein into the expression vector pCAGGS/MCS. pCVSVG was kindly provided by Michael A. Whitt.

HTNV strain 76-118 and SEOV strain SR-11 were used as representative strains of the HTNV and SEOV serotypes, respectively. These strains were propagated in the E6 clone of Vero cells. VSVΔG*G is the recombinant VSV derived from a full-length cDNA clone of the VSV genome (Indiana serotype) in which the coding region of the G protein was replaced by the coding region of the GFP gene and the G protein was expressed in trans (21). VSVΔG*G was kindly provided by Michael A. Whitt.

The expression of G1 and G2 was analyzed by using an indirect immunofluorescent antibody (IFA) test and flow cytometry. 293T cell monolayers in six-well plates (80% confluent) were transfected with 2 μg of pCHTNM and pCSEOM by using Mirus TransIT LT1 transfection reagents (Panvera, Madison, Wis.), as recommended by the manufacturer. After 48 h, cells were examined for protein expression. Cells from six-well plastic plate (Coster) were detached by using a pipette and then suspended in culture medium. They were then centrifuged, and the pellet was resuspended with 1 ml of phosphate-buffered saline (PBS). For intracellular staining, the IFA test was carried out. Briefly, after centrifugation, the pelleted cells were resuspended again with an appropriate volume of PBS. The resuspended cells were spotted onto a 24-well glass slide, air dried, and fixed with acetone for 10 min. Acetone-fixed smears of 293T cells were treated with the culture supernatant from hybridoma clones, producing the monoclonal antibodies (MAbs) 8B6 against G1 and HCO2 against G2, for 1 h at 37°C. After being washed with PBS, fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal antibodies against anti-mouse immunoglobulin G (IgG; Zymed) at 1:400 were added to the well. After 1 h of incubation at 37°C, cells were washed and examined with a fluorescence microscope. For cell surface staining, flow cytometry analysis was done. The transfected 293T cells were detached with a pipette, washed with PBS, and fixed with 3% paraformaldehyde in PBS for 5 min at room temperature. After being washed with 0.5% bovine serum albumin in Dulbecco PBS (fluorescence-activated cell sorting [FACS] solution), the cells were treated with purified MAb (clone HCO2) at 10 μg/100 μl for 30 min on ice. After being washed with FACS solution, the cells were treated with FITC-conjugated anti-mouse IgG (1:100) for 30 min on ice. After another wash with FACS solution, the cells were suspended in 500 μl of FACS solution and analyzed by using a FACSCalibur (Becton Dickinson). The data were evaluated by using Becton Dickinson FACScan research software (CellQuest, v3.0.1.f).

At 36 h after transfection of 293T cells with pCHTNM, pCSEOM, pCVSVG, or pCAGGS/MCS, the cells were infected with VSVΔG*G at a multiplicity of infection of 1 for 1 h at 37°C. The 293T cell monolayer was then washed with 1% heat-inactivated FCS-PBS three times, and culture medium was added. After 24 h of incubation at 37°C in a CO₂ incubator, the culture supernatant was clarified by low-speed centrifugation and stored at −80°C.

For pseudotype virus titration in Vero E6 cells, Vero E6 cells grown on 96-well plates were infected with 50 μl of serially diluted virus stock. After a 1-h adsorption period, the inoculum was removed, fresh culture medium was added, and the cells were incubated at 37°C in a CO₂ incubator. At 16 h postinfection (hpi), the cells were fixed with 1% paraformaldehyde in PBS for 10 min at room temperature, washed with distilled water, and air dried. GFP-expressing cells were counted under a fluorescence microscope. Since pseudotype VSVs are unable to produce infectious progeny virus, the numbers of GFP-positive cells were regarded as infectious units (IU).

The incorporation of the G1 and G2 envelope glycoproteins of HTNV and SEOV into the pseudotype viruses was examined by Western blotting. Culture supernatants of 293T cells were partially purified by ultracentrifugation at 100,000 rpm for 1 h through 20% sucrose in TNE buffer (10 mM Tris, 135 mM NaCl, 2 mM EDTA). The pellets were dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (2% SDS, 10% glycerol, 5% 2-ME, 0.002% bromophenol blue, 0.0625 M Tris-Cl; pH 6.8) for VSV M protein detection or in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.8], 2% Triton X-100, 150 mM NaCl, 600 mM KCl, 5 mM EDTA, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride) for G1 and G2 glycoprotein detection. The virus samples dissolved in SDS-PAGE sample buffer were separated by SDS-PAGE in 12% gels under reducing conditions. The virus samples dissolved in radioimmunoprecipitation assay buffer were separated by PAGE in 8% gels under nonreducing conditions. After electrophoresis, the proteins were blotted onto polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). The membrane was soaked in 3% skim milk in PBS at room temperature to saturate unsaturated areas. For VSV M staining, mouse MAb to M protein (working dilution of 1:1,000; a gift from M. A. Whitt) was used (10). For hantavirus G1 and G2 staining, the undiluted culture supernatants of hybridoma clones of 8B6 against G1 of HTNV and 8E10 against G2 of HTNV were used (2). The membrane was incubated with MAb for 1 h at room temperature. The membrane was then washed three times with 0.05% Tween 20 in PBS for 5 min each and incubated with biotinylated anti-mouse IgG (Zymed) at a 1:5,000 dilution for 1 h. After incubation for 1 h, the membrane was washed according to the same procedure used in the second step and was incubated with horseradish peroxidase-conjugated avidin (1:400; Cappel). The band was detected by using the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech, Ltd.). Briefly, the membrane were treated with a mixture of A solution and B solution (supplied by the manufacturer at a 40:1 volume), incubated for 5 min at room temperature, drained off from the membrane, exposed to Hyperfilm ECL for 15 s, developed with Rendol, and fixed with Renfix.

Clones producing MAbs directed against the HTNV envelope glycoproteins were prepared as previously described (2). Rabbit immune sera against HTNV and SEOV were obtained from rabbits infected with live viruses, as previously described (11). A total of 22 serum specimens from HFRS patients previously diagnosed as being infected by HTNV and SEOV were kindly provided by H. Wang of the Institute of Virology, Chinese Academy of Preventive Medicine, Beijing, China, and 5 specimens were provided by Y. Nishimune of the Research Institute for Microbial Diseases, Osaka University. Ten serum specimens from the United Arab Emirates obtained from patients with high fever of unknown etiology were provided by M. K. Ijaz and T. Al Karmi of the Department of Medical Microbiology, Faculty of Medicine and Health Science, United Arab Emirates, through H.-K. Ooi of the Department of Parasitology, Faculty of Veterinary Medicine, Hokkaido University. These sera were confirmed to be negative for hantavirus infection, as previously reported (24).

The endpoint titers of the neutralizing antibodies were determined by FRNT as described earlier (1). Briefly, sera were serially diluted and mixed with an equal volume containing 30 to 70 focus-forming units of virus per 50 μl. The mixtures were incubated for 1 h at 37°C and then inoculated onto Vero E6 cell monolayers in 96-well tissue culture plates (Iwaki 3860-096; Asahi Technoglass Co., Tokyo, Japan). After adsorption for 1 h at 37°C, the wells were overlaid with medium containing 1.5% carboxymethyl cellulose. After incubation for 7 days in a CO₂ incubator, the monolayers were fixed with acetone-methanol (1:1) and dried. The focus of virus-infected cells was detected by staining the cells with polyclonal serum from a rabbit immunized with truncated N protein (amino acids 1 to 244) from HTNV, followed by the addition of horseradish peroxidase-labeled goat antibodies and substrate. The FRNT titer was expressed as the reciprocal of the highest serum dilution resulting in a reduction in a number of infected cell foci greater than 80%.

A total of 30 μl of medium containing 200 IU of VSV pseudotypes was incubated with an equal volume of serially diluted sample serum for 1 h at 37°C. Then, 50 μl of the mixture was inoculated onto Vero E6 cell monolayers in 96-well tissue culture plates. After adsorption for 1 h, the mixture was replaced with Eagle minimal essential medium. After a 16-h incubation period, the cells were fixed with 2% paraformaldehyde for 10 min, washed with distilled water, air dried, and stored at −80°C until assay. Cells infected with VSV pseudotypes were examined and counted based on GFP expression under a fluorescence microscope.

The cDNA containing the coding region of G1 and G2 of HTNV strain 76-118 and SEOV strain SR-11 was subcloned into the mammalian expression vector, pCAGGS/MCS, which contains a chicken β-actin promoter. We investigated the antigenicity of these recombinant proteins and their cell surface localization. Figure 1A shows representative staining patterns of acetone-fixed 293T cells with MAbs to both G1 and G2 of HTNV and SEOV, which show strong expression and high transfection efficiency of both the G1 and G2 proteins. The recombinant G1 and G2 proteins derived from HTNV and SEOV retained the same reactivity to MAbs as Vero E6 cells infected with the native virus. It has been reported that for the efficient generation of pseudotype VSV by using this system, the transport of envelope glycoproteins to the cell surface is critical (15). Therefore, we also tested the cell surface expression of G1 and G2 by flow cytometry. The expressed envelope glycoproteins were efficiently transported to the cell surface, as indicated in Fig. 1B. The cell surface IFA test also confirmed the surface localization of G1 and G2 (data not shown).

We inoculated 293T cells expressing hantavirus G1 and G2 with the recombinant VSV complemented with G protein (VSVΔG*G), as previously described (18), to examine the incorporation of G1 and G2 proteins into VSV particles. At 30 h after the transfection of pCHTNM, pCSEOM, or pCVSVG, 293T cells were inoculated with VSVΔG*G at a multiplicity of infection of 1. After 24 h, the culture supernatant was harvested and pseudotype viruses were purified by centrifugation through a sucrose cushion (20 and 60%) and analyzed by Western blotting.

M proteins were detected in all of the recombinant VSVs complemented with G protein (Fig. 2, lane 1), the G1 and G2 proteins of HTNV (lane 2), or SEOV (lane 3) by Western blotting analysis after SDS-PAGE separation (Fig. 2A). None of the antibodies that we have tested thus far detects G1 or G2 protein after reducing SDS-PAGE separation, probably because of the highly conformational structure of hantavirus envelope glycoproteins. Therefore, we used a different procedure (native PAGE) to detect G1 and G2 incorporated into each recombinant VSV particle. The smeared, blurred shape of the G1 or G2 bands may be due to the lower performance of native PAGE in separating the proteins compared to SDS-PAGE (Fig. 2B, lanes 2 and 3). In addition, glycosylation of a protein also affects its charge. To dissociate G1 and G2, high concentrations of salt and the nonionic detergent Triton X-100 (2% Triton X-100, 150 mM NaCl, 600 mM KCl) were needed. As a result, surplus chloride ions that reduced the performance of electrophoresis remained in the electrophoresis buffer. These results indicated that G1 and G2 proteins expressed in trans were incorporated into the recombinant VSV particles. The SEOV G1 band was weaker reaction of SEOV G1 compared to HTNV G1. This experiment was performed twice, and both gave a smudged G1 band of the same intensity and pattern. Therefore, the weaker signal of SEOV G1 was due to the heterogeneous reaction of MAb 8B6 produced against HTNV.

To test whether hantavirus G1 and G2 rescued the infectivity of recombinant VSV lacking G protein, we examined the infectivity of VSVΔG*-HTN and -SEO in Vero E6 cells. GFP-expressing cells were first detected 8 hpi. With time, the number of positive cells increased, as did the fluorescence intensity. The number of positive cells plateaued at ca. 16 hpi and then declined. At ca. 23 hpi, the infected cells detached from the dishes. The same growth kinetics were observed with both pseudotype VSVs. The titers of VSVΔG*-HTN and -SEO were ca. 10⁵ to 10⁶ IU/ml and consistently 10-fold higher than those of native HTNV and SEOV produced in Vero E6 cells in three independent experiments performed in our laboratory.

Based on the growth kinetics of the pseudotype VSVs described above, we set the fixation time for the neutralization test at 16 h after inoculation. To confirm the applicability of VSVΔG*-HTN and VSVΔG*-SEO as substitutes for the native virus in the neutralization test, we compared the neutralizing activities of 12 MAbs characterized previously and immune rabbit sera. As shown in Table 1, the MAbs recognizing antigenic sites G1-b, G2-a, and G2-c (3) neutralized the VSVΔG*-HTN infection at the same concentrations as native HTNV. HCO2, which also neutralizes SEOV infection, neutralized VSVΔG*-SEO at the same concentration. A cross-neutralization test with rabbit HTNV and SEOV immune sera showed that VSVΔG*-HTN and VSVΔG*-SEO gave 8- to 32-fold-higher neutralizing titers in response to the homologous antiserum than in response to the heterologous one. This finding is comparable to the results obtained with native viruses (Table 1).

To examine the applicability of the neutralization test for measuring the neutralizing value of human sera and serotyping, we compared the neutralizing titers of the pseudotype VSVs and native viruses by using representative patient serum specimens of each serotype (Fig. 3A). These sera neutralized the native and pseudotype viruses in a dilution factor-dependent manner. The titers based on an 80% reduction of infectivity (neutralizing titer) were almost as high for native viruses as for the pseudotype viruses. The neutralizing titers were roughly two to three times higher for homologous reactions than for heterologous reactions. There was no neutralizing activity with VSVΔG*G. We also took pictures to count the number of GFP-expressing cells in the neutralization test. These showed strong GFP fluorescence, and the number of GFP-positive cells decreased in a dilution factor-dependent manner (Fig. 3B).

Sera from 22 patients previously diagnosed by using the FRNT or IFA test were subjected to the neutralization test with VSVΔG*-HTN, VSVΔG*-SEO, and VSVΔG*G. The neutralizing titers of each virus are shown in Table 2. It was clearly possible to distinguish between HTNV and SEOV infection in human sera with VSVΔG*-HTN and -SEO. All of the sera from healthy individuals and patients suffering from unknown febrile illnesses, which were previously determined to be seronegative for hantavirus infection by the IFA test or the FRNT, were also negative for the pseudotypes VSVΔG*-HTN and -SEO.

Combined with these results, the recombinant VSVs complemented with hantavirus envelope glycoproteins should be useful for measuring neutralizing antibodies and serotyping.

We also tested the stability of the pseudotype VSVs subjected to repeated freezing-thawing, stored at 4°C, and stored at room temperature (25°C). Repeated freezing-thawing (stored at −80°C for 1 year, thawed once, and frozen again) did not affect the infectivity of pseudotype VSVs (data not shown). As shown in Fig. 4, the infectivity titers of the pseudotype VSVs were retained at >10⁵ IU/ml when stored for up to 28 days at 4°C. On the other hand, after 14 days of incubation at room temperature, the infectivity of the pseudotype VSVs decreased to undetectable levels.