Bruce F. Fernie

The Safety and Immunogenicity of a Human Immunodeficiency Virus Type 1 (HIV-1) Recombinant gp160 Candidate Vaccine in Humans

Objective: To evaluate the safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein (rgp160) candidate vaccine in humans. Subjects: Healthy adult...

Analysis of the respiratory syncytial virus fusion protein using monoclonal and polyclonal antibodies.

At least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusion-inhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

Effect of interferon on murine leukaemia virus infection. IV. Formation of non-infectious virus in chronically infected cells.

Interferon (150 units/ml) was used to treat SC-1 and AKR-2B cells which were chronically infected with murine leukaemia virus (MuLV). This led to a 100-fold decrease in the amount of infectious virus released into the medium and a 10-fold decrease in the number of virus particles measured by the virion-associated reverse transcriptase assay. However, there was little change in the amount of cell-associated infectious virus, though nearly twice as many cell-associated virions were counted in electron micrographs. With both types of cells, interferon blocked MuLV replication at the post-budding stage, but it did not change the morphology of the particles produced or their content of virion 70S RNA. Infectious virus assembled on the cell membranes of interferon-treated cells was less stable at 37 degrees C than that grown in the absence of interferon. Release of infectious virus from interferon-treated cells was not inhibited by actinomycin D or cycloheximide, though both agents inhibited virus production in controls. These results show that interferon inhibits MuLV replication through effects on virion assembly; these lead both to the formation of non-infectious particles and of fewer virions. Kinetic analysis further shows that interferon affects MuLV assembly rapidly and induction of an antiviral protein may not be required.

The Development of Balb/c Cells Persistently Infected with Respiratory Syncytial Virus: Presence of Ribonucleoprotein on the Cell Surface

Abstract Balb/c mouse cells were persistently infected with RS virus. Continued passage of the virus produced in these Balb/c cells resulted in a 10-fold increase in the amount of infectious virus produced. Approximately 80% of the BCH4 cells expressed RNP and “spike” antigen intracellularly and associated with the cell surface.

Classification of Hybridomas to Respiratory Syncytial Virus Glycoproteins

Abstract We have classified 28 hybridomas to the RS virus glycoproteins, VP66, VP84, by virus neutralization, RIP, and RIA tests. Without resorting to RIP, a combination of neutralization tests and RIA on BCH4 and RS/HO cells could be used to classify 79% of the hybridomas antibodies correctly. VP66 contains major determinants for virus neutralization since 11/13 hybridoma antibodies to this protein neutralized RS virus while 0/15 hybridoma antibodies to VP84 neutralized the virus.

The stabilization and purification of respiratory syncytial virus using MgSO4

Abstract Respiratory syncytial (RS) virus infectivity is stabilized by MgSO 4 . In the presence of 1 M MgSO 4 at 4°, only 50% of viral infectivity is lost in 12 weeks. This is a 30-fold increase in RS virus stability. The increased stability of RS virus allowed infectivity to be used as a convenient marker for the presence of RS virus during multiple cycles of density gradient centrifugation.

Monoclonal antibodies to respiratory syncytial virus: Detection of virus neutralization and other antigen-antibody systems using infected human and murine cells

Monoclonal antibodies to human respiratory syncytial (RS) virus-specific antigens can be obtained without preliminary recourse to large-scale culture and purification of the virion. Lytically infected human and persistently infected murine cultured cells expressing RS virus-specific cell surface and cytoplasmic antigens were substituted as priming immunogens and as substrates in solid-phase antibody radioimmunoassays. Seven hybridoma clones secreting murine IgG of either the gamma 1 or the gamma 2a subclass bearing kappa light chains were isolated. Two of the antibodies were specific for cell surface viral antigens, but only one was able to neutralize RS virus infectivity. The five remaining antibodies did not neutralize virus infectivity and were specific for viral antigens associated with large cytoplasmic inclusions as judged by indirect immunofluorescence (IF) analysis on fixed infected cells. Similar IF analysis using live cells revealed that those antigens, associated with the cytoplasmic inclusions in both the human and murine infected cells, were not expressed on the cell surface of the live infected human cells, but were expressed on the cell surface of the live infected murine cells. Monoclonal antibodies generated via the present system will prove useful in the immunological analysis of viral components which are specific pathogenic functions, such as infectivity, and those which may be abnormally exposed at the surface of persistently infected cells.

Kinetics of Synthesis of Respiratory Syncytial Virus Glycoproteins

The synthesis of the two respiratory syncytial (RS) virus glycoproteins (VP66 and VP84) was examined under standard conditions and after treatment with tunicamycin and monensin. The protein backbone for VP66, the fusion protein (F1,2) is cotranslationally glycosylated to form F0, which is cleaved to form F1,2 by 20 min of chase. Monensin treatment inhibited the cleavage of F0 over an 80 min chase period, indicating that this occurred late in the transit of F0 through the Golgi apparatus or after exit from the Golgi apparatus. Tunicamycin treatment resulted in the synthesis of a 50K to 55K unglycosylated F0 which is cleaved to a 40K protein. VP84, the large glycoprotein, contains a protein backbone of only 26K to 30K which is modified by N-linked and probable O-linked glycosylation. Tunicamycin treatment results in the synthesis of a 70K protein (p70) which incorporates [3H]glucosamine and [3H]fucose but not [3H]mannose. Glycosylated precursors varying in mol. wt. from 29K to 45K (p45) are found in infected cells at regular 2K to 3K intervals, producing a ladder effect. The step from p45 to VP84 is severely delayed by monensin treatment thereby enhancing the ladder effect of the precursors.

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles (interferon virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but interferon virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in interferon virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted interferon virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.

Monoclonal antibodies for the rapid diagnosis of respiratory syncytial virus infection by immunofluorescence

Eight separate monoclonal antibodies to the Long strain of respiratory syncytial virus (RSV) were tested for their utility as rapid diagnostic reagents in immunofluorescence. Preliminary screening indicated that all 8 reacted with 11 field strains from three previous local RSV outbreaks and with 4 of 5 additional strains chosen because of their antigenic diversity by neutralization. Two monoclonal antibodies, one each directed against a surface glycoprotein and the nucleocapsid protein, were then compared, singly and combined, with a polyclonal antiserum as diagnostic reagents in 209 consecutive samples submitted to our diagnostic laboratory. Agreement between the two monoclonal antibodies was 100% and between them and the polyclonal serum was 98%. Sensitivity in relation to culture was 96-98%. Monoclonal antibodies are excellent immunofluorescent diagnostic reagents; antigenic diversity among RSV strains was not an impediment to their use in this study.