Neutralizing and enhancing activities of human respiratory syncytial virus-specific antibodies.

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.

In vitro enhancement of respiratory syncytial virus infection of U937 cells by human sera.

Human 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.

Immunoblot analysis of the human antibody response to respiratory syncytial virus infection.

The protein specificities of the antibodies induced during natural respiratory syncytial (RS) virus infection in humans were investigated using immunoblotting of human sera against virus proteins resolved by gel electrophoresis. Sera from 33 patients, who had complement-fixing antibodies against RS virus, were analysed. All the patients showed a response against the virus nucleoprotein (VPN41) and glycoprotein (VGP48). Antibodies against the virus proteins GP90, VPP32, VPM27 and two proteins of molecular weights 24,800 and 23,700 were also demonstrated. The spectrum of protein specificities of the antibodies varied among patients. There was an increase in the level of antibody for these virus proteins between the acute and convalescent serum samples from five patients. The protein specificities of the IgG and IgM antibodies were determined for five patients; VPN41, VPP32 and VPM27 induced IgG and IgM in all of these sera. Two of the five patients had detectable IgG but not IgM antibodies against VGP48, whereas the remaining three sera had detectable VGP48-specific IgG and IgM antibodies.

Antigenic variation between human respiratory syncytial virus isolates.

Three 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.

Monoclonal antibodies to human respiratory syncytial virus and their use in comparison of different virus isolates.

Monoclonal 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 phospho-protein 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.

Analysis of polypeptides in Tacaribe virus-infected cells.

Two virus-induced polypeptides designated p79 (mol wt, 79,000) and p105 (mol wt, 105,000) in BHK21 or Vero cells infected with Tacaribe (Tac) virus have been identified. Both polypeptides were found in immune precipitates with antiserum to Tac virus, suggesting that they are virus specific. Two-dimensional gel analysis of Tac virus-infected Vero cell extracts indicated that p79 and p105 were acidic proteins which did not comigrate with any polypeptides from uninfected cells. Neither of the polypeptides was found to be phosphorylated under conditions in which phosphorylation of the N (nucleocapsid) protein was detected. Comparison of one-dimensional peptide maps of the p79 polypeptide and the nucleoprotein indicated that they are unrelated in primary sequences.

Oligonucleotide fingerprint analysis of Tacaribe virion RNA.

Polyacrylamide gel electrophoretic analysis of RNA segments of the arenaviruses Pichinde (Pic) and Tacaribe (Tac) showed them to be distinguishable in that Pic S RNA had a slower electrophoretic mobility than Tac S RNA. The L and S RNA segments of Tac virions were found to have distinct RNase T1 oligonucleotide fingerprints, indicating that they are unique RNA species. The oligonucleotide patterns of the Tac L and S RNAs were also distinct from those of the corresponding Pic RNA segments.

Antigen and polypeptide synthesis by temperature-sensitive mutants of respiratory syncytial virus.

A 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 GP1 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 of 39 degrees C. Mutants ts 17 (complementation group B') and ts 19 (group E) were almost completely restricted, suggesting defective early functions. Mutants ts A1 (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 1 (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 degrees C. Absence of surface filaments in ts mutant-infected cells at 39 degrees C confirmed their virus-specific nature.

Seven complementation groups of respiratory syncytial virus temperature-sensitive mutants.

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. (P. F. Wright, M. A. Gharpure, D. S. Hodes, and R. M. Chanock, Arch. Gesamte Virusforsch, 41:238--247). Thus seven complementation groups of respiratory syncytial virus, designated A, B, C, D, E, F, and G, have been defined. The frequency and type of mutant isolated varied according to strain; group C was unique to the A2 strain, and groups D, E, F, and G were unique to the RSN-2 strain. The highest complementation indexes were obtained by preincubation for 7 h at permissive temperature, followed by incubation at restrictive temperature for 40 to 50 h in the case of A2 strain mutants or 80 to 90 h for RSN-2 strain mutants. Genetic recombination was not detected.