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Arch Virol (1992) 122:107-118

~) Springer-Verlag1992 Printed in Austria

Antibody-dependent enhancement of hantavirus infection in macrophage cell lines J.-S. Yao t, H. Kariwa 1, I. Takashima 1, K. Yoshimatsu 2, J. Arikawa 2, and N. Hashimoto 1 1Department of Veterinary Public Health, Faculty of Veterinary Medicine, and 2 Laboratory of Animal Experiment, Institute of Immunological Science, Hokkaido University, Sapporo, Japan

Accepted May 20, 1991

Summary. Antibody-dependent enhancement (ADE) of hantavirus infections (strains Hantaan 76-118 and SR-11) was studied using macrophage-like cell lines (J774.1, P388D1, and U937). Significantly higher virus titers (1,000 to 4,000 FFU/ml) were obtained by pretreatment of the virus with immune serum as compared to normal serum (< 20 FFU/ml). Monoclonal antibodies (MAbs) to strain Hantaan 76-118 were employed to determine the antigenic determinants responsible for the ADE activity. ADE of the infection occurred with MAbs to both G1 and G2 envelope glycoproteins, but not with MAbs to nucleocapsid protein. Antigenic determinants related to haemagglutination or virus neutralization were found to cause ADE of the infection.

Introduction Haemorrhagic fever with renal syndrome (HFRS) is a general term for disease caused by members of the genus Hantavirus, family Bunyaviridae, with high mortality [29]. This disease, characterized by renal disorder, fever and haemorrhagic manifestations, was reported in Korea, China, the Soviet Union, and in northern and eastern Europe [-14]. In particular, 50,000 to 100,000 patients are still reported in China annually [10, 25]. Recently, various wild rodent species were reported to have antibody to hantavirus [12]. Furthermore, evidence shows that hantavirus has already spread worldwide, including areas where no patients were reported [6, 7, 13, 16, 28]. Infected rodents carry the virus persistently and are believed to be a source of infection of humans [2]. Inactivated vaccines prepared from infected mouse or rat brain tissues or tissue culture cells are under development in Korea, China, and Japan [26, 30, 31, 32]. In addition, recombinant component vaccines using baculovirus or vaccinia virus as expression vectors have been developed [22].

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It has been reported that virus infection of particular cells, those bearing receptors for the Fc portion of immunoglobulin, is enhanced under the presence of antiviral antibody [9, 19]. This phenomenon, so-called antibody-dependent enhancement (ADE) of viral infection, might be caused by the binding of the virus and the antibody complex to the Fc receptor on the macrophage. Dengue shock syndrome or dengue haemorrhagic fever (DSS/DHF) and the early deaths of mice experimentally infected with rabies virus are suspected to be the result of A D E mediated by a pre-existing antibody to a different type of dengue virus and inadequate immunization against the rabies virus, respectively [3, 8, 21]. Yamanishi etal. [18] reported that rodent macrophages were susceptible to hantavirus infection and replication, and played an important role in the spread of virus infection in vivo. Recently, Nagai [17] elucidated the A D E of infection of hantavirus in a macrophage-like Cell line and in peripheral and splenic macrophages from the nude mouse passively transferred with MAb to envelope glycoprotein after virus challenge, and pointed out its significance for the development of vaccine. For further understanding of the role of antibody in hantavirus infection, we established an in vitro system using three different macrophage-like cell lines originating from mice or humans. A panel of monoclonal antibodies (MAbs) to envelope protein of hantavirus was used to determine the antigenic sites related to ADE. Materials and methods

Cells Murine macrophage-like cell lines J774,1 and P388D1, and human macrophage-like cell line U937 (kindly provided by Dr. K. Hattori, Hokkaido University and Dr. J. KimuraKuroda, Tokyo Metropolitan Institute for Neuroscience) were used. These cells were grown in RPMI 1640 (Gibco, Grand Island, N.Y., U.S.A.) containing 10% fetal calf serum (FCS), 50gM 2-mercaptoethanol and antibiotics (100 units/ml of penicillin and 100gg/ml of streptomycin). Vero E6 cells were grown in Eagle's minimum essential medium (MEM, Nissui Co., Tokyo, Japan) supplemented with L-glutamine (0.292 g/l) and 5% FCS. Viruses and antisera Hantavirus strains Hantaan 76-118 and SR- 11 [11, 15], which were isolated from Apodemus agrarius and laboratory Wistar rats, respectively, were used. The viruses were propagated in a monolayer of Vero E6 cells as previously described, and culture fluid was harvested and stored at -80 °C as a stock virus until used. Antisera to strains Hantaan 76-118 and SR-11 were prepared in rabbits (provided by Dr. J. M. Dalrymple of USAMRIID, Fort Detrick, U.S.A.). A panel of MAbs directed to G1 and G2 envelope glycoproteins of Hantaan 76-118 was prepared as previously described [1]. Preparation of Fab fragment of anti-Hantaan IgG MAb 1G8 was purified with protein A-sepharose column (Pharmacia, Uppsala, Sweden). Preparation of Fab fragment was carried out by papain digestion according to the methods

ADE of hantavirus infection

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reported by Casale et al. [5]. Briefly, purified IgG was digested with papain (enzyme : protein ratio of 1 : 400), Fab fragment was obtained by passing through protein A-sepharose column. Protein concentrations and IFA titers were estimated spectrophotometrically by absorbance at 280 nm and with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-F(ab')2 of mouse IgG respectively.

ADE experiment Approximately 5 × 104 macrophage-like cells were distributed into wells of 96-well tissue culture plates (Falcon, Lincoln Park, New Jersey, U.S.A.) and incubated at 37 °C in a humidified 5% CO2 incubator. The next day, polyclonal antibodies or MAbs were serially diluted tenfold in the dilution medium (Eagle's MEM with 5% FCS, pH 7.4) and each diluted antibody was mixed with an equal volume of hantavirus for 30 rain at 37 °C. Normal rabbit serum or mouse ascitic fluid was used as a control. The antibody and virus mixtures were inoculated into macrophage-like cell monolayers at a final multiplicity of infection (m.o.i.) of 0.05. After incubation for 1 h at 37 °C, the mixtures were removed and RPMI 1640 with 10% FCS containing no antibodies was added to the cell monolayers. Four days after incubation at 37 °C in a 5% CO2 incubator, culture fluid or cells were harvested for titration of infectivity. The ADE titer was defined as the reciprocal of the highest antibody dilution exhibiting an infectivity titer of 20 FFU/ml or greater in culture fluid. The virus titer in culture fluid was measured by counting the number of infectious foci by the peroxidase-anti-peroxidase (PAP) method as described by Tanishita et al. [-273. Briefly, culture fluid from each dilution was inoculated into a monolayer of Vero E6 cells grown in 96-well tissue culture plates. After incubation for 1 h at 37 °C, the inoculum was removed and overlay medium (Eagle's MEM containing 1.5% carboxymethyl cellulose and 5% FCS) was added to each well. On day 6, the overlay medium was removed and the cell monolayers were washed four times with 0.05 M phosphate-buffered saline (PBS, pH 7.4) and fixed with 100% methanol at room temperature for 15min. After being dried, antiSR rabbit serum (50 ~1, 1 : 300) was added to each welt, after which anti-rabbit IgG sheep serum (50 gl, 1 : 300) was also added. Next, the PAP complex (50 gl, 1 : 1000) was added to the wells. Between each step, the cell monolayers were washed four times with PBS. Finally, substrate solution, composed of 3 mg of 3,3-diaminobenzidine tetrahydrochloride and 0.1 ml of 3% H202 in 10ml PBS for color development, was added to each well and the number of foci was counted under a dissection microscope. The number of infected macrophage-like cells was measured by infectious center assay (ICA). Briefly, macrophage-like cells were suspended in RPMI 1640 with 10% FCS, and suspensions were inoculated onto Vero E6 cell monolayers grown in 24-well tissue culture plates (Falcon, Lincoln Park, New Jersey, U.S.A.). After incubation overnight at 37 °C, the inoculum was removed and the monolayers were overlaid with the overlay medium. On day 6, the number of infectious centers was counted by the PAP technique as described above.

Detection of viral antigen The indirect immunofluorescence antibody (IFA) test was used for detecting viral antigen in infected macrophage-like cells. The cell suspensions were seeded onto multitest slides and incubated for 2h at 37°C. Then the cells on the slides were fixed with 100% cold acetone for 15 rain i n an ice bath and anti-SR rat serum was added. After incubation for 1 h at 37 °C, 'the slides were washed three times with PBS and fluorescein isothiocyanate (FITC)-conjugated goat antibody to rat IgG was applied. After further incubation, the slides were washed and observed under an immunofluorescence microscope. The number of cells showing specific immunofluorescence was counted.

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J.-S. Yao etal. Results

Antibody-dependent enhancement (ADE) of infection mediated by polyclonal antisera To examine the susceptibility of macrophage-like cell lines to hantavirus infection, J774.1 cells were inoculated with strain Hantaan 76-118 or SR-11 at a final m.o.i, of 0.05. On day four, culture fluid and cells were harvested for assay. Although the virus titer of the infected culture fluid was under the detection level ( < 20 FFU/mt), some culture cells were shown to be infected with strain Hantaan 76-118 or SR-11 by the IC assay and IFA test (Table 1). The number of infected cells was approximately 80/105 cells for Hantaan 76118 and 28/105 cells for SR-11. Therefore, J774.1 cells were proved to be susceptible to hantavirus infection, although the infection efficiency was very low. Next, the infection of J774.1 cells with hantavirus was examined in the presence of polyclonal antisera. As shown in Table 2, when the J774.1 cells were infected with strain Hantaan 76-118 or SR-11 mixed with various dilutions of homologous antiserum, neutralization was seen with high antiserum concentrations (10-l to 10-2). However, with further dilution of the antiserum, enhancement of the infection appeared and the peak of enhancement (1,780 FFU/ ml for Hantaan 76-118 and 760FFU/ml for SR-11) was shown in the 10 - 3 dilution of homologous antiserum. On the other hand, in the case of J774.1 cells infected with the mixture of virus and heterologous antiserum, the cross enhancement of infection was also observed and the peak enhancement (2,080 FFU/ml for Hantaan 76-118 and 680FFU/ml for SR-11) was seen in the 10 .2 dilution, which was 10-fold lower than that of the virus homologous antiserum combination. Similar results were also obtained by counting the number of infected cells by the infectious center assay (Table 2). However, no enhancement was observed when using normal rabbit serum as a control. To ascertain that the ADE of hantavirus infection is a general phenomenon, other macrophage-like cell lines P388D1 (derived from mice) and U937 (derived from humans) were used for ADE experiments. Strain Hantaan 76-118 was Table 1. Infection of J774.1 cells

HTN SR-tl

Virus titer~ of culture fluid (FFU/ml)

No. of infected cells ICA (FFU/105 cells)

IFA (%)

< 20 < 20

80 28

0.1 < 0.1

The virus titer of the culture fluid was titrated by the PAP method

ICA Infectious center assay IFA Indirect immunofluorescenceantibody test

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Table 2. Antibody-dependent enhancement of infection of strains Hantaan 76-118 and SR11 in J774.1 cells Antiserum

Dilution (log10)

No. of infected cells (10s)a

Virus titer of culture fluid (FFU/ml) b

HTN

SR-11

HTN

SR-11

Anti-HTNc rabbit

- 1 - 2 - 3 - 4 -5

Antibody-dependent enhancement of hantavirus infection in macrophage cell lines.

Antibody-dependent enhancement (ADE) of hantavirus infections (strains Hantaan 76-118 and SR-11) was studied using macrophage-like cell lines (J774.1,...
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