key: cord-0004973-yii5txf1 authors: Arikawa, J.; Yao, J. -S.; Yoshimatsu, K.; Takashima, I.; Hashimoto, N. title: Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies date: 1992 journal: Arch Virol DOI: 10.1007/bf01309700 sha: db9c57c002f1b80a59b1af24a88559fe18380fd1 doc_id: 4973 cord_uid: yii5txf1 To investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2±2.1 to 21.5±2.8 days) being significantly shorter than that of the control group (25.8±1.8, p<0.01, Mann-Whitney,U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the antivirus antibody. Haemorrhagic fever with renal syndrome (HFRS) is a rodent borne viral disease characterized by fever, renal disorder and hemorrhagic manifestations [34, 35] . HRFS patients have been reported throughout Eurasia and parts of Africa [12] particularly in China [8, 27] , Korea [11] , Northern [10] , and Eastern Europe [15] . Hantaan virus is the causative agent of HFRS and classified in the family Bunyaviridae, genus Hantavirus [24] . This virus has two different glycoprotein projections (G1 and G2) on the surface of the virion [25] . In our previous Vero E6 cells infected with Hantaan virus, strain 76-118 were monodispersed in culture medium and dropped onto spot slide glasses. They were incubated at 37 °C in a CO2 incubator for four hours to extend the cells on the glass, then washed with PBS. The cells were fixed with cold acetone and used as antigen. Fluorescent isothiocyanate (FITC) conjugated anti-mouse immunoglobulins or anti mouse IgM (~t chain specific) (goat; Cappel Laboratories, Cochranvilla, Pa.) were used as the second antibody. IFA titers were expressed as the reciprocals of the highest dilution giving specific immunofluorescence in the infected cell cytoplasm. IFA titers of 1 : 10 or more were regarded as positive [3] . FRNT was described previously [203. Briefly, a serial 10-fold dilution of mouse ascitic fluid or immune serum was mixed with an equal volume of virus suspension. The mixture was kept at 37 °C for 1 hr, then inoculated onto Veto E6 cell monolayers in 96 well plates. After incubation for 1 hr, the inoculum was removed and the monolayers were covered with overlay medium (MEM containing 1.5% carboxymethylcelullose and 5% FCS). After incubation for 7 days at 37 °C in a CO2 incubator, the cell monotayer was washed with phosphate buffered saline (PBS) and fixed with methanol for 10 min at room temperature. Infected cell foci were stained by the peroxidase-anti-peroxidase (PAP) method with rabbit immune sera and the number of infected cell foci was counted. FRNT titer was regarded as the highest dilution of ascitic fluid causing 80% or more reduction in the number of infected foci formed in the presence of control ascitic fluid instead of antibody. In the focus inhibition neutralization test (FINT), a virus suspension containing 30 to 50 focus-forming units (FFU) of virus per 50 gl was inoculated onto Vero cell monolayers in 96 well plates. After incubation for 1 hr, the inoculum was removed and the cells were covered with overlay medium containing serial 10-fold dilutions of ascitic fluid. The FINT titer was defined by the 80% reduction method as described for the FRNT titer. Outbred ICR mice were obtained from Shizuoka Laboratories (Shizuoka, Japan) and mated to produce suckling mice. A group of 8 to 10 suckling mice (less than 24 hr after birth) were inoculated subcutaneously (s.c.) with 50 gl of undiluted ascitic fluid containing a MAb specific for Hantaan virus, strain 76-t 18 (Table t) or normal ascitic fluid. Four hours after the adoptive transfer of the ascitic fluid, mice were challenged with a s.c. injection of 5 × 103 FFU (10 LDs0) of Hantaan virus, strain 76-118. Survival rates were recorded for 5 weeks after the challenge. Serum specimens of surviving mice were obtained by cardiac puncture under ether anesthesia. The filter paper method [-3] was used for collecting blood from dead or moribund mice. The passive protection study was carried out one time to each MAb clones, except to clone 6D4. All animals were treated according to the Laboratory Animal Control Guidelines in our institute which was basically in conformity to National Institutes of Health-American Association of Laboratory Animal Control Guidelines. All the animal experiments were carried out in a class P3 facility. Organs were removed from mice when they were moribund, dead, or 35 days post challenge (surviving mice). Each organ was homogenized as a 10% suspension in Eagle's MEM containing 5% fetal calf serum, 60 ~tg/ml of kanamycin, 400 units/ml of penicillin, and 400 ~tg/ml of streptomycin. Ten-fold dilutions of the suspension were inoculated onto Vero E6 cell monolayers in 96 well plates. After incubation for 1 hr at 37 °C, the inoculum was removed and replaced with overlay medium. After incubation for 7 days, infectivity titers were measured by the PAP method as described previously [-30] . Anti H a n t a a n + + + + + + + + mouse immunesera a _ Less than 80% focus reduction or inhibition at 1 : 10 dilution; m o r e than 80% focus reduction or inhibition at 1" 10 ( + ) , 1" 100 ( + + ) , 1 : 1,000 ( + + + ) dilution and 1C6 to antigenic site G2-f however, were negative by FRNT, but inhibited focus formation. Thus, additional neutralizing antigenic sites were found out by FINT. Expression of viral antigens on the surface of infected cells was examined by the membrane FA test. As listed in Table 1 , all the MAb clones reacted with unfixed infected cells. The intensity of FA was similar regardless of the MAb clones used. To examine the antigenic sites related to protective immunity in suckling mice, at least one MAb clone representative of each of the different antigenic sites was selected and passively transferred to suckling mice followed by a lethal Anti Hantaan --mouse immunesera Medium 5.08 4.90 -alogl0 FFU/g tissue b Less than 200 FFU/g tissue challenge with H a n t a a n virus, strain 76-118 (Table2). All the F R N T M A b s (clones 16D2, HCO2, 16E6 and 11El0) completely prevented lethal infection. In addition, clones 16D2, H C O 2 and 11El0 protected all or half o f the mice from infection since they h a d no sero-conversion after the challenge. Clone 8E10, positive in F I N T but negative in F R N T , protected all the mice from lethality but failed to protect from infection. Thus, either G1 or G2 envelope proteins were responsible for induction of protective immunity. Interestingly, clone 6D4 to antigenic site Gl-a-2, which had no neutralizing activity in either F R N T or F I N T , protected all the mice from lethal infection. The experiment was repeated and the same result was obtained. The rest of the non-neutralizing clones were divided into two groups depending on the protective activity from lethal infection. Most of the mice which received M A b clones 8B6, 20D3, 23G10-1 and G D O 5 died, but clones EBO6 and 17G6 partially protected the mice from lethal infection. As described above, non-neutralizing M A b s failed to protect the mice from lethal infection. M e a n time to death for mice are listed in the right h a n d column of Table 2 . Although the survival rates differed between clones transferred, the mean time to death among groups of mice ranged from 18.2 4-2.1 (clone 8B6) to 21.5 + 2.8 days (clone 23Gt0-1) and were significantly shorter than those of the control group (25.8 4-1.8, p < 0.01, Mann-Whitney U probability test). Survival curves for the mice transferred with the non-protective antibodies (clones GDO5, 8B6 and 20D3) are shown in Fig. 1 a. In all groups, mice began to die 4 to 5 days earlier than control mice. On the other hand, though clone 17G6 or EBO6 partially protected the mice from lethal infection, they began to die earlier than the controls (Fig. 1 b) . Virus titers in brain, lung and spleen tissues of immunized mice were measured and compared with those of the control group. Organs were collected when mice were moribund, dead, or 35 days after the challenge (surviving mice). Virus was recovered from the organs from dead mice, but not from those of the survivors. Higher virus titers for dead mice were detected in brain tissues rather than in the tung or spleen. Virus titers obtained from brain tissues were about ten times higher than those from control mouse brain, although the virus titers in lung tissues were similar for control and immunized mice. One of the G1 antigenic sites and two of the G2 sites of Hantaan virus envelope protein are involved in virus neutralization, as demonstrated by plaque reduction neutralization with a panel of MAbs [1] . In the present study, an additional antigenic site related to virus neutralization was identified with MAb clones 8El0 and 1C6, which were positive in FINT but negative in FRNT (Table 1 ). In paramyxoviruses, when the MAb to fusion (F) protein was contained in the agar overlay medium, plaque formation was effectively inhibited comparing to its plaque reduction neutralization activity [31] . The inhibition of plaque formation is considered to be caused by the inhibition of cell to cell spread of virus infection, since the (fusion) F protein mediated the cell to cell fusion but not relate to the virus attachment to cell. We have reported that Hantaan virus has cell fusion activity underthe low pH condition [2] . Therefore, to examine the neutralization mechanism related to the newly recognized antigenic site, attachment blocking activity as well as fusion inhibition activity of clones 8El0 and 1 C6 will be required. To investigate the role of Hantaan virus envelope protein in infection in vivo, suckling mice were passively given MAbs followed by Hantaan virus challenge. All the animals transferred with FRNT MAbs either to G1 or G2 protein were completely protected from Hantaan virus challenge because all the mice survived and most of them showed no sero-conversion (Table 2) . These results were consistent with a recent report by Schmaljohn et al. [23] , in which hamsters were passively protected from infection only with MAbs having plaque reduction neutralizing activity. In addition, MAb clone 8El 0, which recognized the antigenic site related to neutralization discovered here, also protected suckling mice against lethal infection, although it did not protect the mice from infection ( Table 2 ). The MAb may retard the spread of viral infection in mice. The fatal outcome of experimentally inoculated mice or rats was strictly dependent on the age of the animals. Complete resistance from lethal Hantaan virus challenge was gained at two weeks of age in mice [9, t 8] and at one week in the rat [37] . Therefore, the delay in viral spread at an early stage of infection may result in survival at a later stage due to age-dependent resistance. These results confirmed the important role of envelope protein as protective antigen. Clone 6D4 to antigenic site Gl-a-2 protected all the mice from lethal infection, although the antibody had no neutralizing activity in either FRNT or FINT and also had no HAI activity. The protective effect of non-neutralizing antibody in passively transferred mice has been reported in various viruses [7, 14,.20-22, 26, 29] . All the reports ascribed the protective mechanisms to cell mediated immune responses against infected cells, such as complement-mediated lysis of infected cells, or antibody dependent cell-mediated cytotoxicity (ADCC). Since this MAb clone was positive in the membrane FA test but lacked complement binding activity (subclass IgGt), ADCC rather than complement-mediated cytolysis of infected cells was suggested. No protective activity was detected in the remaining non-neutralizing clones for reasons which remain to be determined. To examine relationship between this phenomenon and antigenic site (Gl-a-2) in more detail, we are now planing to carry out similar experiments with the other MAb clone 10Fll, which binds to the same antigenic site as clone 6D4. As shown in Fig. 1 and Table 2 , mice began to die earlier than the control group and the mean time to death were significantly shorter than those of control mice. Virus titers in the brains of mice that died early 117.5 x 105 to 1.5 x 106FFU/g), were apparently higher than those of control mice (1.2 x 105 FFU/g), although the virus titers in the lungs were similar. Several animal experiments have indicated that the death of challenged mice is closely related to virus titers in brain tissues and virus titers of a lethal threshold are around 105FFU per gram [17, 18, 28] . Recently, the antibody dependent enhancement (ADE) of hantavirus infection to Fc-receptor bearing cells such as macrophages, caused by enhanced binding of virus and antibody complex via Fc receptors on the cell has been reported [36] . Since macrophages are susceptible to hantavirus infection and are believed to be responsible for the spread of infection in mice [ 16] , ADE of infection to macrophages is considered a plausible mechanism for the rapid growth of virus in the brain. To examine if the early death presented here is related to high virus titers in the brain, studies on the kinetics of virus growth in various organs of mice with or without antibody transfer are now in progress. Similar phenomena have been reported in mice challenged with yellow fever and JE [5] , Langat [32] and rabies viruses [19] , as well as cat coronavirus [33] , but few reports have discussed the mechanisms involved. Gould et al. [6] reported that enhanced neurovirulence in mice infected with yellow fever virus is not mediated through Fc and complement receptor-bearing macrophages, but that the cytotoxicity to infected brain tissues is caused by antibodies. 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I. The effect of the administration of specific antiserum Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever Hemorrhagic fever with renal syndrome: memorandum from a WHO meeting Hemorrhagic fever with renal syndrome: Global epidemiology and ecology of hantavirus infections Antibody-dependent enhancement of hantavirus infection in macrophage cell lines Comparison of virulence between two strains of Rattus serotype hemorrhagic fever with renal syndrome (HFRS) virus in newborn rats This study was supported in part by Grants-in-Aid for Scientific Research 0'~o. 02806056) and for Development Scientific Research (No. 03558009) from the Ministry of Education, Science and Culture of Japan. Authors' address: Dr. J. Arikawa, Institute of Immunological Science, Hokkaido University, Sapporo 060, Japan.Received September 10, 1991