ABSTRACT
We have produced a genetically-engineered chimeric protein composed of the external domains of the respiratory syncytial virus (RSV) fusion (F) protein and the parainfluenza virus type 3 (PIV-3) hemagglutinin-neuraminidase (HN) protein in insect cells using the baculovirus expression system. The yield of the soluble chimeric FRSV-HNPIV-3 protein could be increased approximately 2-fold by using Trichoplasia ni (High Five) insect cells in place of Spodoptera frugiperda (Sf9) for expression. The chimeric protein, purified from the supernatant of baculovirus-infected High Five cells by immunoaffinity chromatography was correctly processed at the F2-F1 proteolytic cleavage site. Immunochemical analysis of the chimera with a panel of anti-F and anti-HN monoclonal antibodies suggested that the antigenicity of the major F and HN neutralization epitopes of the chimeric protein was preserved. Immunization of cotton rats with two 1 or 10 micrograms doses of the chimeric protein adsorbed to aluminum phosphate elicited strong PIV-3 specific HAI responses as well as PIV-3 and RSV specific neutralizing antibodies, and at either dose completely protected against challenge with live RSV and PIV-3.
Subject(s)
HN Protein , Parainfluenza Virus 3, Human/immunology , Recombinant Fusion Proteins/immunology , Respiratory Syncytial Viruses/immunology , Vaccines, Synthetic , Viral Vaccines , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Baculoviridae/genetics , Base Sequence , Gene Expression , Gene Transfer Techniques , Genetic Engineering , Hemagglutinins/chemistry , Hemagglutinins/genetics , Hemagglutinins/immunology , Molecular Sequence Data , Moths/metabolism , Neuraminidase/chemistry , Neuraminidase/genetics , Neuraminidase/immunology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Viral Envelope Proteins , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
Allantoic fluids harvested from embryonated chicken eggs infected with reference strains of influenza A viruses were analysed for subtilisin inhibitor activity. While all acid heat-treated and nontreated virus-infected fluids could reduce subtilisin activity, fluids of FM and Bangkok strains had the greatest inhibitory ability. The degree of subtilisin inhibition closely paralleled the appearance of infectious Bangkok and FM virus in allantoic fluid. Maximum levels were achieved at 48 hr post-infection (p.i.) Ultracentrifugation analyses indicated that the bulk of thermostable inhibitor(s) of 48 hr Bangkok and FM infectious fluids remained in the supernatant rather than sedimenting with the viral pellet.
Subject(s)
Influenza A virus/analysis , Subtilisins/antagonists & inhibitors , Allantois/metabolism , Allantois/microbiology , Animals , Chick Embryo , Influenza A virus/metabolism , Viral Proteins/metabolismABSTRACT
Neutral protease activity of allantoic fluid from embryonated chicken eggs was quantified during the course of influenza virus infection. Antigenic subtypes of influenza A viruses selected for study were H1N1 strains PR/8/34, Brazil/8/78, FM/1/47, the H3N2 strain Bangkok/1/80 and the H5N9 Turkey/ /Ontario/66 as well as the Sendai strain of parainfluenza type 1 virus. Three different types of profiles of allantoic fluid proteases could be readily distinguished after infection of eggs with various virus strains. In all profiles, periodic peaks of protease activity always preceded the partial shut down of protamine cleaving proteases which paralleled the production of near maximum titers of infectious virus. To determine the mechanism involved in this reduction of proteolytic activity, infectious allantoic fluids were analysed for the presence of protease inhibitors. Acid heat treated 48 hour virus-infected allantoic fluids of different influenza strains could inhibit the activities of subtilisin and allantoic fluid proteolytic enzymes.
Subject(s)
Allantois/enzymology , Extraembryonic Membranes/enzymology , Influenza A virus/physiology , Orthomyxoviridae Infections/enzymology , Peptide Hydrolases/metabolism , Animals , Chick Embryo , Hot Temperature , Hydrogen-Ion Concentration , Orthomyxoviridae Infections/microbiology , Parainfluenza Virus 1, Human/physiology , Paramyxoviridae Infections/enzymology , Paramyxoviridae Infections/microbiology , Protease Inhibitors/analysis , Virus ReplicationABSTRACT
The levels of neutral protease activity associated with allantoic and amniotic fluids of embryonated eggs during the replication of influenza strains A/PR/8/34 (H1N1) and A/turkey/Ontario/7732/66 (H5N9) were investigated. A sensitive fluorometric technique proved useful for characterization and monitoring changes of protease activities in egg fluids. The predominant type of protease in allantoic and amniotic fluids had trypsin-like specificities. Variation in protease levels of both fluids occurred throughout the course of virus replication irrespective of the virus strain or the route of inoculation used. Concomitant with the production of high levels of infectious virus there was a marked decrease in neutral protease activity in the fluid from the cavity initially infected. Translocation of virus also occurred especially with amniotically infected eggs, as evidenced by high infectious virus titers and decreased protease activities in allantoic fluids.
Subject(s)
Allantois/enzymology , Amniotic Fluid/enzymology , Extraembryonic Membranes/enzymology , Influenza A virus/physiology , Peptide Hydrolases/metabolism , Virus Replication , Allantois/microbiology , Amniotic Fluid/microbiology , Animals , Chick Embryo , Protease Inhibitors/pharmacology , TurkeysABSTRACT
The proteolytic susceptibility of polypeptides of four antigenically distinct subtypes of influenza a virus strains of human origin was studied. The extent of degradation of polypeptide molecules of strains A/PR/8/34 (H0N1) (PR), A/FM/1/47 (H1N1), A/Singapore/1/57 (H2N2) and A/Hong Kong/8/68 (H3N2), assessed by densitometry of gels after sodium dodecylsulfate polyacrylamide gel electrophoresis was variable by treatment with trypsin. Also, sequential treatment of PR strain initially with phospholipase D followed by proteases of different specificities suggested differences in susceptibility of surface and internal polypeptide molecules. The significance of these results is discussed.
