SUMMARYThree hybridoma antibodies, prepared against the RSN-2 strain of human respiratory syncytial (RS) virus, have been used to identify antigenic variation between 41 isolates of RS virus collected from widely separated geographical regions over a period of 29 years. One antibody was directed against an antigenic site on the virus fusion protein, VP70. This site was shared by 21 virus isolates tested and its recognition by the antibody was sensitive to the presence of 2-mercaptoethanol. The remaining two antibodies used react against the virus phosphoprotein, VPP32. Two independent sites were recognized on VPP32 by these antibodies. One antibody reacted with all of the virus isolates screened while the second reacted with only 21 out of the 41 virus isolates. On the basis of the variable epitope, two antigenic types of human RS virus were identified. The distribution of each antigenic group among 28 RS virus isolates from the Grampian Region, north-east Scotland, collected between 1982 and 1984 was determined. The reactivity of these antibodies was examined using immunofluorescence staining and by immunoblotting; the latter technique also revealed that the electrophoretic mobility of VPP32 varied in parallel with the variable antigenic site.

SUMMARYMonoclonal antibodies to the RSN-2 isolate of human respiratory syncytial (RS) virus have been characterized with regard to their specificity for viral polypeptides and for different RS virus isolates. Five hybridoma antibodies recognized the phosphoprotein VPP32 and the other recognized the matrix protein VPM27. Evidence was obtained to support the view that VPP32 was associated with the nucleoprotein VPN41. Three of the antibodies to VPP32 showed cytoplasmic immunofluorescent staining while the other two showed only surface staining of virus-infected cells. The immunoblot technique was used to determine the immunoreactivity of four of the hybridoma antibodies against human RS isolates other than RSN-2. One of these antibodies reacted exclusively with RSN-2 virus isolate whereas the others detected determinants shared by all human RS isolates tested. Extension of this approach may offer the possibility of typing RS isolates using monoclonal antibodies.

SUMMARYA revised nomenclature for the polypeptides of respiratory syncytial (RS) virus has been devised on the basis of comparison of the Long, A2 and RSN-2 strains by slab-gel electrophoresis. Seven polypeptides, now designated VP200, VGP48, VPN41, VPP32, VPM27, VP25 and VP10, were observed in preparations of all three strains of RS virus, irrespective of the host cell of origin. In addition, a slowly migrating glycopolypeptide GP 1 was prominent in partially purified RS virus of the Long and A2 strains obtained from Hep-2 cells, and to a lesser extent from BS-C-1 cells. In the case of the RSN-2 strain, this polypeptide was only resolved clearly in virus obtained from Hep-2 cells. GP1 was an atypical glycopolypeptide in that 35S-methionine incorporation was poor relative to 3H-glucosamine incorporation.The ts mutants of RS virus exhibited four distinct phenotypes with respect to intracellular polypeptide synthesis and antigen production at 39 °C. Mutants ts 17 (complementation group B') and ts 19 (group E) were almost completely restricted, suggesting defective early functions. Mutants ts A 1 (group A), ts A7 (group C) and ts 1 (group D) synthesized antigen and polypeptides normally, but the amount of antigen at the cell surface was reduced, suggesting maturation defects. In addition, the VPP32 of ts l (group D) exhibited an aberrant mobility, confirming its viral specificity. The remaining mutants, representing groups B, F and G exhibited generally impaired synthesis at 39 °C.Absence of surface filaments in ts mutant-infected cells at 39 °C confirmed their virus-specific nature.

SUMMARYHuman sera containing respiratory syncytial (RS) virus-specific antibodies enhance RS virus infection of the U937 macrophage cell line. There was an increase in the number of cells expressing virus antigen when U937 cells were infected with RS virus in the presence of human serum compared to cells infected in the absence of human serum. Human sera enhanced virus yield, as measured by the cell-released infectious virus, by an average of 50-fold compared to virus infection in the absence of human serum. The comparison of the enhancing activities of paired acute and convalescent human sera showed that the titre of enhancing antibody increased in parallel with the titre of RS virus-specific antibody measured by complement fixation and virus neutralization. An RS virus-specific neutralizing monoclonal antibody directed to the virus F protein enhanced virus infection of U937 cells. A non-neutralizing monoclonal antibody directed to the virus nucleoprotein did not enhance virus infection. The possible role of enhancing antibodies in vivo is discussed. INTRODUCTIONRespiratory syncytial (RS) virus is a major cause of acute respiratory infections in young children. The use of a formalin-inactivated RS virus vaccine predisposed children to severe illness when subsequently infected with RS virus (Kim et al., 1969). Following immunization, children developed lower neutralizing antibody titres than those of a comparable age who had natural RS virus infections and had not been immunized (Murphy et al., 1986). Chin et al. (1969) found higher virus shedding and virus isolation rates in vaccinees compared to unvaccinated children when both groups were naturally infected with RS virus. These data led to the speculation that low antibody titres contributed to the severity of the RS virus infections. A possible explanation for these data is antibody-dependent enhancement of virus infection, as proposed by Porterfield (1982) and Halstead (1982), although no experimental data were presented at that time.The in vitro enhancement of virus infection of macrophage cell lines by virus-specific antibodies at sub-neutralizing concentrations has been reported for several viruses (reviewed by Halstead, 1982). The initial stages of virus enhancement require the formation of a virusantibody complex which then attaches, via the Fc portion of the antibody, to the macrophage Fc receptor; this facilitates the entry of the virus as compared to virus infection in the absence of antibody (Gollins & Porterfield, 1984). The sites of replication of RS virus in the respiratory tract are poorly understood and the role of antibody-dependent enhancement in RS virus pathogenesis remains to be determined. Recently, RS virus has been shown to replicate in vitro in human peripheral blood mononuclear leukocytes (Krilov et al., 1987). In addition, RS virus antigen has been found in circulating mononuclear leukocytes of infants with RS virus infections (Dumorat et al., 1985). The present report describes the infection of the U937 human macrophage cell line by...

The neutralizing and enhancing activities of respiratory syncytial virus (RSV)-specific antibodies were examined. These two biological activities were measured for a panel of six monoclonal antibodies (MAbs) specific to the RSV surface F and G glycoproteins. Four MAbs specific for the F protein possessed both neutralizing and enhancing activities. One MAb (11-2-D2), specific to the G protein, enhanced RSV infection of U937 cells, a human macrophage cell line, but did not neutralize virus infectivity. One MAb (11-3-A3), specific to the F protein, efficiently neutralized virus infectivity but did not enhance RSV infection of U937 cells. MAb 11-3-A3 neutralized representative strains of the two antigenic subtypes of RSV. Assays performed with mixtures of MAbs showed that high concentrations of MAb 11-3-A3 masked the enhancing activity of MAb 11-2-D2. The assay of mixtures of two MAbs possessing only enhancing activities demonstrated that this response was synergistic. The role of neutralizing and enhancing antibodies in determining the outcome of RSV infection was examined for infants from whom cord blood serum samples were collected at birth. There was no significant difference in the magnitude of the serum-enhancing activities between infants who were hospitalized with RSV infections and a group of age-and sex-matched control infants with no reported respiratory illness requiring hospitalization. However, the results indicated a possible correlation between RSV infection of the infants and the occurrence of in vitro antibody-dependent enhancement of the cord blood sera at a serum dilution of 10 ؊2. A significant inverse correlation was found between the plaque-neutralizing and enhancing activities of the cord blood sera from infants, irrespective of subsequent RSV infection. These data are discussed in relation to the possible contribution of antibody-dependent enhancement to the normal course of RSV pathology in vivo.

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Fifteen temperature-sensitive mutants of the RSN-2 strain of respiratory syncytial virus have been classified into six complementation groups, two of which appeared to be homologous with two of the three complementation groups of the A2 strain described by Wright et al.