A. C. R. Samson

The purification of four respiratory syncytial virus proteins and their evaluation as protective agents against experimental infection in BALB/c mice.

The fusion (F) glycoprotein, large glyco- (G) protein, phospho- (P) protein and 22K protein of respiratory syncytial (RS) virus A2 strain were purified by a combination of immunoaffinity adsorption and preparative SDS-PAGE. All four proteins elicited serum antibody in mice after repeated inoculation in adjuvant, although the magnitude of the response as measured by ELISA varied from mouse to mouse. The F protein generated neutralizing antibodies in only 50% of the mice determined to be seropositive by ELISA. The G protein also induced neutralizing antibodies but in this instance neutralization tests and ELISA titres were more closely correlated. No neutralizing activity was detected in mice immunized with the P or 22K proteins although all produced antibody detectable by ELISA. Mice immunized with either the F or the G protein were found to be protected against subsequent RS virus challenge, whether they had developed neutralizing antibody or not. Mice inoculated with the P or 22K proteins were not protected.

Location of neutralizing epitopes on the fusion protein of Newcastle disease virus strain Beaudette C

A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.

Strain Variation of Respiratory Syncytial Virus

Western blotting and immunoperoxidase staining with monoclonal antibodies were employed to analyse epitopic and polypeptide molecular weight variation among isolates of respiratory syncytial (RS) virus collected in Newcastle between 1965 and 1986. One group of isolates resembled the A2 and Long prototype subgroup A strains of RS virus in possessing a P protein of Mr 34,000. Isolates in this subgroup showed two patterns of reactivity with subgroup A-specific monoclonal antibodies to the G glycoprotein and 22K protein. Isolates with both reactivity patterns were isolated throughout the period studied. Isolates in the second group resembled the 8/60 subgroup B prototype strain in their lack of reactivity to subgroup A-specific monoclonal antibodies but were heterogeneous in P protein molecular weight. The earliest isolate only, made in 1965, possessed a P protein of Mr 31,000 resembling the prototype strain. All subsequent subgroup B isolates possessed a higher Mr, 33,000, P protein. Overall, subgroup A viruses were isolated most frequently although subgroup B strains may have predominated in some epidemics.

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the alpha-peptide of beta-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.

A neutralizing monoclonal antibody to respiratory syncytial virus which binds to both F1 and F2 components of the fusion protein

A virus-neutralizing monoclonal antibody (1E3) specifically immunoprecipitated the 70000 mol. wt. (70K) fusion (F) protein from respiratory syncytial (RS) virus-infected HeLa cells. Western blotting analysis of polypeptides from such cells separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that 1E3 was peculiar in that it bound to both F1 (50K) and F2 (20K) components of the F protein. Antibody subsequently eluted from either the F1 or the F2 regions of immunoblots re-bound to both F1 and F2 regions of the SDS-PAGE blot. These results show that monoclonal antibody 1E3 reacts with an epitope which is found on both F1 and F2 subunits of RS virus fusion protein.

Heterogeneity of the respiratory syncytial virus 22K protein revealed by Western blotting with monoclonal antibodies

Respiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23,000 in our gel system). Three novel polypeptides with mol. wt. of 24,000, 21,000 and 17,000 were stained in addition to the 23,000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under non-reducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24,000, 21,000 and 17,000 polypeptides were not visible, the number of mobility variants of the 23,000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23,000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.

Anomalous behaviour of Newcastle disease virus haemagglutinin-neuraminidase protein in Western blotting analysis of monoclonal antibody binding sites.

Previous studies with a particular monoclonal antibody (MAb 445) raised against the Ulster strain of Newcastle disease virus (NDV) have shown that this MAb immunoprecipitates only the 74,000 mol. wt. (74K) haemagglutinin-neuraminidase protein (HN) of both Ulster and Beaudette C strains of NDV. Using the technique of Western blotting, it is shown that under certain denaturing conditions virion proteins comigrating in SDS-polyacrylamide gels with the faster migrating 67K fusion protein and 53K nucleocapsid/nucleocapsid-associated protein, as well as the 74K HN, all reacted strongly with MAb 445 and with MAb 14 which is also directed against the HN protein. Analysis of this anomalous behaviour using SDS-polyacrylamide diagonal gel electrophoresis has led to the unexpected conclusion that the 74K HN can electrophorese as three immunoreactive species of apparent mol. wt. 74K, 67K and 53K. If the Western blotting technique is to be applied to proteins which have not been sufficiently denatured (in an attempt to preserve epitope integrity) it is important to establish that no additional proteins remain associated with the protein bands that react with the MAb.

Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells

Immunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h post-infection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.

Respiratory syncytial virus glycoprotein expression in human and simian cell lines.

Glycoproteins synthesized in both human (HeLa and HEp-2) and simian (Vero and BS-C-1) cell lines following infection with two different strains of respiratory syncytial virus (A2 and Long) were analysed by SDS-PAGE following immunoprecipitation with monoclonal antibodies. Minor virus strain-dependent differences in the large glycoprotein, G, and the fusion protein polypeptides F1 and F2 were observed together with minor cell line-dependent differences in the size of the F2 polypeptide. Major quantities of two glycoproteins, termed Ga (50K) and Gb (45K), were detected in A2 strain-, and to a lesser extent in Long strain-, infected simian cells. These proteins were also present in infected human cells, but in much reduced amounts. Immunoprecipitation with anti-G monoclonal antibodies demonstrated that Ga and Gb shared different epitopes with G.

Identification of Haemagglutinin-Neuraminidase Antibody Binding Sites by Western Blot Analysis of Antibody-resistant Mutants and Partial Digest Fragments of Newcastle Disease Virus

A collection of monoclonal antibodies (MAbs) which react with the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) has been used to isolate MAb-resistant mutants of the Beaudette C strain of NDV. The patterns of cross-reactivity of the HN proteins of these mutants against the collection of MAbs determined by Western blotting allowed the MAbs to be sorted into different groups. Protease V8 partial digest fragments of purified wild-type virions and subsequent reaction against the collection of MAbs using Western blotting provided an alternative method of grouping MAbs which broadly agreed with the former method. Chemical cleavage of the HN protein at aspartate-proline bonds followed by Western blotting of the fragments allowed the approximate position of certain MAb binding sites to be determined.