key: cord-1000044-m8r0tpof authors: Corbeil, Serge; Seguin, Cecile; Trudel, Michel title: Involvement of the complement system in the protection of mice from challenge with respiratory syncytial virus Long strain following passive immunization with monoclonal antibody 18A2B2 date: 1996-04-30 journal: Vaccine DOI: 10.1016/0264-410x(95)00222-m sha: b131952702a5f7e4a5e30e8c384943262e77c0db doc_id: 1000044 cord_uid: m8r0tpof Abstract Passive immunization of mice with 131 μg of the non-neutralizing monoclonal antibody (mAb) 18A2B2, directed against the A subgroup epitope of the G glycoprotein of respiratory syncytial virus Long strain (RSV), confers protection against viral i.n. challenge. The role of the Fc fragment of this antibody as well as the involvement of antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis towards protection was evaluated in vivo. Passive immunization with the Fab fragment alone (618–907 μg mouse−1) was unable to confer protection in mice. Furthermore, we passively immunized with the mAb 18A2B2 SCID beige mice, which are deficient in natural killer (NK) cell activity, to ascertain the role of NK cells in the protective mechanism. These mice were free of virus 5 days following viral challenge, indicating that NK cells do not contribute significantly towards the protective action of this antibody. Moreover, passively immunized BALB c mice decomplemented with 8–10 U of cobra venom factor (CoVF) and DBA 2J mice (C5 deficient) were only partially protected. These findings suggest that in mice the alternative and classical pathways of the complement system are involved in the passive protection mechanism conferred by the non-neutralizing mAb 18A2B2. To our knowledge, it is the first description of a protective mechanism in mice that involves a non-neutralizing antibody and the complement system. Respiratory syncytial virus causes severe lower respiratory tract disease in infants and children',*. For several years the F and G glycoproteins of RSV have been under scrutiny in the prospect of developing an effective and innocuous vaccine1g5. Studies involving the characterization of potential antigenic and immunogenic sites of the F and G glycoproteins revealed that some B epitopes are involved in neutralization and passive protection . 3~6-11 It was shown that the subgroup specific antigenic site is located within the amino acid region 174-188 of the glycoprotein G839. Furthermore, we previously demonstrated" that a synthetic peptide consisting of the amino acids 174-187 conferred protection in BALB/c mice from intranasal challenge with the RSV, Long strain. Passive immunization with mAb 18A2B2, *Centre de Recherche en Virologie, lnstitut Armand-Frappier, Universitb du Quebec, 531 boul. des Prairies, Laval, Quebec, Canada H7N 423. tTo whom correspondence should be addressed. (Received 17 July 1995; revised 3 October 1995; accepted 4 October 1995) directed against the above epitope, also mediates an antiviral effect in vivo but has no neutralizing activity in vitro". These observations suggest that the Fc portion of the mAb 18A2B2 may play a critical role towards protection in vivo either through an ADCC response' '12, a complement-mediated cytolysis or a complement-dependent virolysis' 3~1 7. In this study we examined the mechanisms involved in the protection of mice following passive immunization with mAb 18A2B2. The protective potential of the mAb was investigated in SCID mice deficient in B and T cell function, in SCID beige mice deficient in B, T, NK cells and polymorphonuclear (PMN) cells. Furthermore, complement depleted BALB/C mice and DBA/2J mice deficient in the C5 component of the complement were used to evaluate the role of the complement system in protection. Finally, IFN-y depleted BALB/c mice allowed us to evaluate the contribution of IFN-), towards the antiviral effect observed in BALB/c mice passively immunized with mAb 18A2B2. The results demonstrate that both pathways of the complement Protection of mice: S. Corbeil et al. system are involved in the protection of mice passively immunized with the non-neutralizing mAb 18A2B2 directed against the A subgroup epitope of glycoprotein G of RSV, Long strain. BALB/c mice were purchased from Charles River Laboratory (St-Constant, Qc.) SCID and SCID Beige mice were purchased from Charles River Laboratory (Wilmington, MA) and the DBA/2J mice (C deficient strain, lacking C5) from Jackson Laboratory (Bar Harbor, ME). All mice were female, 2840 days old. The Long strain of respiratory syncytial virus (ATCC VR-26; American Type Culture Collection, Rockville, MD) was propagated on Hep-2 cells (ATCC CCL-23) previously grown in equal parts of Eagle's minimal essential medium and medium 199 (GIBCO Laboratories, Grand Island, NY), supplemented with 50 pug ml -' of gentamicyn and 5% (v/v) fetal calf serum (FCS). When infected, monolayers were incubated in serum-free medium. Virus titrations were carried out in 24-well, flatbottom microtiter plates on HEP-2 cells (4 x lo4 cells well ~ '). The medium from confluent cultures was removed and 200 ~1 from a tenfold dilution of virus was added to the wells. In the case of virus obtained from lung extracts, the solution was added as a tenfold dilution. After a 90 min adsorption period, 1.5 ml of 1% serum medium were added to each well. Plates were incubated for 5-10 days at 37°C in a humidified atmosphere containing 5% CO,. Viral syncytia were then counted: titers were expressed as tissue culture infectious doses (log,, TCID,,). Purification of mAb 18A2B2 was carried out by standard Protein-A-Sepharose chromatography". Fab fragments of mAb 18A2B2 were obtained by papain digestion. Briefly, 10 mg of purified mAb 18A2B2 were digested in 0.1 M acetate and 3 mM ethylenediaminetetraacetic acid (EDTA) buffer, pH 5.5, with 0.5 mg papain (Sigma Chemical Co., St Louis, MO) preactivated with 50 mM cysteine (Sigma) for 7 h at 37°C. The reaction mixture was then passed twice through a protein-A-Sepharose column to eliminate Fc fragments and undigested antibodies. Purified Fab fragments were dialyzed against phosphate buffer saline solution (PBS) (Gibco), pH 7.4, and stored at -20°C until use. The purity of the fragments was evaluated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and Coomassie Blue staining", followed by densitometry with a GS-670 laser densitometer (Bio-Rad, Mississauga, Ontario). The activities of mAb and Fab fragments were determined by ELISA. In passive protection studies, groups of mice were immunized intravenously (i.v.) with 131 pg of mAb 18A2B2 and were immediately challenged intranasally (i.n.) with 50~1 of lo'.' TCID,, RSV, Long strain. Five days following challenge the mice were killed and their lungs were tested for the presence of virus by titration assay. Complement depletion was achieved by injection of Baja nuja cobra venom factor (CoVF) (Sigma). Each mouse received 8 doses of 1.0 or 1.25 U'O using the following schedule and route of inoculation, where challenge occurs at time 0: -24 h, -19 h, 0, +6 h via i.p. route and +24 h, +48 h, +72 h and +96 h postchallenge via i.v. route. At time 0 mice received 13 1 pug of mAb 18A2B2 (i.v.) and were challenged as described previously. In all cases described above, the animals were killed 5 days after challenge and their lungs were tested for the presence of virus by titration assay. Depletion of IFN-y was acheived by injection of anti-IFN-y antibodies (CedarLane Laboratories, Horby, Ontario) via i.v. route. Each mouse received 300 ,ug of antibodies 2 h before challenge. In addition each mouse received 131 ,ug of mAb 18A2B2 i.v. 30 min before challenge. The animals were killed 5 days after challenge and their lungs were tested for the presence of virus by titration assay. Three hundred nanograms per well of RSV or control Hep-2 antigen purified by sucrose density step gradient centrifugation (30/50%, w/v) were adsorbed overnight, on flat-bottom microtiter plates at 4°C. The plates were washed, blocked for 30 min with PBS containing 10% FCS (Gibco Laboratories, Grand Island, NY) (v/v). MAb 18A2B2 and Fab fragments serially diluted in PBS supplemented with 10% FCS and 0.02% Tween 20, were added. After 2 h incubation at 20°C the plates were washed and 100 ~1 of a l/1000 dilution of horseradish peroxidase-labeled rabbit anti-mouse IgG (Cappel, Scarborough, Ontario) or goat anti-mouse IgG (Fab-specific) antibodies (Sigma) were added to each well. Plates were further incubated for 2 h at 20°C washed again and the reaction developed with O-phenylenediamine [0.4 mg ml -' in 1 M sodium citrate, pH 5.0 containing 0.03% (v/v) HzO,]. Results were expressed as the inverse of the last serum dilution that gave a reading above the average reading plus two standard deviations of an isotypic mAb control. Neutralization assays were carried out in microtiter plates: 25 ~1 of mAb 18A2B2 (131 ,&lqO ~1) were incubated overnight in the presence or absence of 2.5 ~1 of mouse complement (Sigma). To the mixture was then added and mixed 25 ~1 of viral suspension containing 100 TCID,O ml -' of RSV, Long strain. The neutralization reaction was allowed to proceed for 4 h at 4°C. The virus-antibodies mixtures were added to plates containing Hep-2 cell sheets, incubated at 37°C for 90 min before the addition of 150 ,ul of 1% (w/v) methylcellulose semisolid culture medium. After an incubation period of Protection of mice: S. Cofbeil et al. In order to characterize the protective mechanism conferred to mice by 131 ,ug of mAb 18A2B2, we passively immunized BALB/c mice with either this mAb or with its purified Fab fragment, then challenged the mice with RSV, Long strain. MAb 18A2B2 prevented replication of RSV in the lungs of treated mice (Table I) . However, mice receiving 618-907 ,ug of the Fab fragments showed no significant reduction of viral titres in the lungs (t-test 0.05 level) in comparison to the control mice receiving intranasal challenge only (Table 1) . This observation is consistant with the observation made by Oldstone et aL2' that Fab fragments are almost totally ineffective in the lysis of viral infected cells. Lamarre and Talbot22 showed that the Fab fragment of a neutralizing mAb directed against the S glycoprotein of murine hepatitis virus required tenfold the amount of the whole antibody to achieve the same level of protection in mice. In our experiment, the complete absence of protection observed with the passive transfer of up to 907 pug of Fab fragments suggests that the Fc fragment of the n-&b 18A2B2 plays an important role in passive protection. Unfortunately, we have not been able to evaluate the protective potential of antibody bivalency by using F(ab'), since the isotype IgG2b is degraded upon digestion with pepsin. SCID mice have few if any lymphocytes; they are hypogammaglobulinaemic and deficient for immune functions mediated by T and B lymphocytes23q24. We immunized SCID mice with mAb 18A2B2 to determine whether B and T cells play a role in the protection conferred. The 6 mice receiving mAb were totally protected while the control mice had an average viral titer of 104.3 TCID,,. Total protection observed in passively immunized SCID mice suggests that passive protection by mAb 18A2B2 is not dependent on B-or T-cell functions. Previous studies reported that mice expressing the beige mutation (bg/bg) have an NK defect which appears to lie within the lytic machinery of NK cells2'-' . The use of beige mice has proven valuable in studying cytomegalovirus and lymphocytic choriomeningitis virus infection in the absence of functional NK cells28.29. Our previous observation that the presence of Fc fragments may be required for protection of mice brought forward the possibility that NK cells could be involved via an ADCC mechanism. In order to test this hypothesis we used the beige mouse model that would allow in viva abrogation of NK cell activity. In our studies the total protection of the SCID beige mice passively immunized with mAb 18A2B2 (Table 2 ) suggests that NK cell lysis of virus-infected cells does not contribute significantly towards the protective action of the mAb 18A2B2. Cobra venom factor is known for its anticomplementary effects in vitro and in vim, particularly on the third and fifth components of the complement system20g30. Moreover, the role of both the alternative and classical pathways of the complement system in host recovery from viral infection has been documented. Oldstone et al. ls showed that C3-dependent crosslinking of polyoma viral particles and the presence of C3b on the viral surface play a significant role in the neutralization of this virus. The importance of C3 in the clearance of polyoma-antibody complexes was shown by delayed removal of the complexes from the serum after in vivo We carried out an experiment using CoVF to ascertain whether inhibition of the classical and the alternative pathway would affect the immune protection observed in mice passively immunized with mAb 18A2B2. BALB/c mice treated with CoVF had an average viral titer of 103." TCID,, in their lungs which was approx. lOOO-fold higher than the average titer (~10 '-~ TCID,,) of mice passively immunized with mAb 18A2B2 (Table I) . However, complement-depleted mice had an average viral titer lower than mice of the control group not passively immunized nor complement-depleted ( 104.74 TCID,,). Our results show that BALB/c mice depleted of complement by CoVF treatments are not protected following viral challenge. Since the inactivator complex CoVF affects not only C3 but also C5 in mice2', our findings strongly suggest that at least one pathway of the complement system is involved in the observed protection of mice by mAb 18A2B2. Our data are consistent with the results obtained by Hirsch et aZ.31 who showed that lOOO-fold more virus was present in the brains of complement-depleted mice. Similar observations on the role of the complement system were made by Hicks et al.32 concerning complement depleted mice undergoing more severe infections with influenza viruses in comparison to normal mice. In order to clarify the role of the classical pathway alone in the protection, we used DBA/2J mice deficient in C5 component of the complement system. Table 3 shows that out of six DBA/U mice passively immunized with mAb 18A2B2, only two were protected. The other four mice had a low average viral lung titer of 1O2.49 TCID,, while control mice not passively immunized with the mAb showed a high average viral titer of 104." TCID,,. In comparison to mice depleted of complement by CoVF treatments, the CS-deficient mice had lower viral titers in their lungs (t-test, P10.01). These data seem to indicate that the classical pathway characterized by the action of C5 is involved to a lesser extent in the protection. The infected cells. These results suggest that the complement system may show different mechanisms of action with respect to the type of mAb involved such as neutralizing or non-neutralizing. It was shown that administration of anti-IFN-y serum enhanced the mortality of mice infected with herpes simplex virus"' and raised virus titers of lymphocytic choriomeningitis virus34 and vaccinia virus35 infected organs. In our study, we attempted to evaluate the effect of IFN-y as a complementary mechanism in the protection of mice. Our results show that three mice injected with anti-IFN-y antibodies and passively immunized with mAb 18A2B2 were totally protected as were the mice passively immunized with mAb 18A2B2 that were not IFN-), depleted (Table I) . This finding suggests that the participation of IFN-y in the observed protection with mAb 18A2B2 is unlikely. This conclusion is also supported by the fact that we were unable to detect any IFN-y in the sera of mice vaccinated with the peptide G/174-187 (result not shown). Our study demonstrates that the complement system is partially involved in the protection of mice passively immunized with mAb 18A2B2 and challenged with RSV, Long strain. However, depletion of the complement system appears not to be sufficient to totally abrogate the protection of mice passively immunized with mAb 18A2B2. Macrophages and monocytes known notably for their viral phagocytic properties and cytokines production could confer a complementary protection in our animal model. This hypothesis is yet to be verified. 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