Monoclonal Antibodies to Human Respiratory Syncytial Virus and Their Use in Comparison of Different Virus Isolates

SUMMARYDifferences in the properties of homologous intracellular structural components of eight strains of subtype A and eight strains of subtype B of human respiratory syncytial (RS) virus were examined. The size of the fusion (F) protein cleavage products and the phosphoprotein (P) showed systematic differences between virus strains representing the two subtypes. The apparent mol. wt. in SDS-polyacrylamide gels under reducing conditions was 48000 (48K) and 46K to 47K for the cleavage product FI in subtype A and B strains, respectively. The size of the F2 protein was 18K to 20K. The subtype B strains showed a slightly higher tool. wt. of this protein compared to the subtype A strains. The size of the P protein was 36K in subtype A strains, but only 34K in subtype B strains. Variations also occurred in the size of the glycoprotein (G) and the 22K to 24K structural protein. These variations did not correlate with the virus subtypes, but were strain-specific. The size of non-glycosylated forms of the F protein cleavage products was determined by use of material from tunicamycin-treated cells. A 44K to 45K non-glycosylated form of the F 1 protein was detected with subtype A virus strains, but the corresponding protein of subtype B strains was not reproducibly identified, presumably due to instability in the absence of glycosylation or altered antigenicity. Monoclonal antibody immunosorbent-bound viral glycoproteins were partially digested with proteases. The pattern of breakdown products of the F1 protein was distinctly different between subtype A and B strains, but it was similar among strains of the same subtype. No subtype-specific pattern was seen in proteolytic digests of monoclonal antibody-bound G protein. INTRODUCTIONFor the 3 decades since its discovery human respiratory syncytial (RS) virus has been considered to represent a single homogeneous serotype. Although cross-neutralization tests with animal hyperimmune sera demonstrated differences between isolates of the virus (Coates et al., 1963; Wulffet al., 1964;Doggett & Taylor-Robinson, 1964) no corresponding differences were observed with infant sera and they were therefore not considered to be of any relevance. With the advent of monoclonal antibodies (MAbs) the antigenic stability of RS virus could be assessed again. Studies with such reagents revealed that isolates of RS virus exhibited variations in epitope occurrence (Gimenez et al., 1984) but that these variations were more extensive than could be explained by incidental mutational changes (Anderson et al., 1985;Mufson et al., 1985). The isolates could be separated into two categories based on their characteristic reaction pattern with different MAbs. The two groups were named RS virus subtype A and B (Mufson et al., 1985). These subtypes showed different properties in at least four different structural proteins emphasizing that they had become ecologically adjusted after a period of separate evolution. Characterization of the epidemiological occurrence of the two RS virus subtypes

Section: E N O R R B Y M a M U F S O N A N D H S H E S H B E R mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural Differences between Subtype A and B Strains of Respiratory Syncytial Virus

Norrby

Mufson

Sheshberadaran

1986

“…Cytocentrifuge preparations were made from the cell suspensions, fixed with acetone at -20 °C for 1 min and stained using bovine anti-RS virus and anti-bovine FITC-conjugated sera (Wellcome) (Gimenez et al, 1984).…”

confidence: 99%

In vitro Enhancement of Respiratory Syncytial Virus Infection of U937 Cells by Human Sera

Gimenez

Keir

Cash

1989

Self Cite

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...

“…Recent studies using monoclonal antibodies to human RSV have identified two (Gimenez et al, 1984;Stott et al, 1984a;Ward et al, 1984;Mufson et al, 1985) or three (Anderson et al, 1985) distinct antigenic groups. While these respective authors examined many different virus isolates, the studies concur in grouping the Long and A2 strains (with other early strains) separately from the 8/60, 18537 and RSN-2 strains, although Anderson et al (1985) were able to define further antigenic differences between the A2 (and A1) strains and the Long strain.…”

Section: Introductionmentioning

confidence: 99%

Murine Cytotoxic T Cells Specific to Respiratory Syncytial Virus Recognize Different Antigenic Subtypes of the Virus

Bangham¹,

Askonas²

1986

SUMMARYThe aim of this study was to establish whether cytotoxic T cells (Tc), raised against respiratory syncytial virus (RSV) in the mouse, are specific to the strain of immunizing virus, or cross-reactive between virus strains. Several recent studies using monoclonal antibodies have begun to define the antigenic variation among strains of RSV. It is likely that a successful RSV vaccine will need to contain antigenic determinants from more than one subtype, but since only the highest levels of neutralizing antibody are able to give complete protection against RSV infection, a vaccine may also need to elicit a cellular immune response. We have recently described H-2-restricted, RSVspecific Tc following RSV infection in the mouse; we present here evidence that polyclonal RSV-specific Tc in the mouse recognize syngeneic target cells infected with every human strain of RSV tested, whatever the subtype. The only RSV strain that appeared not to be recognized was bovine RSV, which seems unable to infect mouse cells; however, bovine cells, infected with bovine RSV and fixed with glutaraldehyde, primed mice for Tc which recognized human strains of RSV.