Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and the elderly. Efforts to develop satisfactory live or inactivated vaccines have not yet been proven successful. Our research focuses on the development of four purified live attenuated RSV sub-type A human vaccine clones. Temperature sensitive (ts) and attenuated purified clones of either cold-adapted (ca) RSV or high-passage (hp) RSV were administered intra-nasally (i.n.) to BALB/c mice and tested for immunogenicity. All four clones produced significant anti-RSV F IgG2a and IgG1 titres in the sera of mice, RSV-specific neutralizing titres higher than those produced by their wild-type progenitor viruses, cytotoxic T-lymphocyte (CTL) activity, and total protection against wild-type (wt) viral challenge. These purified vaccine candidates await testing in humans to determine which contain the required balance between immunogenicity and attenuation.

Protection against respiratory syncytial virus infection by DNA immunization.

Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon gamma after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit beta-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

A prototype recombinant vaccine against respiratory syncytial virus and parainfluenza virus type 3.

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.

Comparative analysis of the immunoprotective abilities of glycosylated and deglycosylated parainfluenza virus type 3 surface glycoproteins.

The role of carbohydrate moieties on the immunoprotective ability of parainfluenza virus type 3 (PIV-3) haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins was tested in hamsters. HN and F proteins were purified from detergent-solubilized virus by lentil-lectin affinity chromatography and deglycosylated by treatment with endoglycosidase F (endo F). Immunization of hamsters with either 1 or 5 micrograms of mock-treated (glycosylated) affinity-purified proteins elicited strong haemagglutination inhibition and neutralizing antibody responses 4 weeks after the primary injection. In contrast, titres were significantly lower with endo F-treated (deglycosylated) proteins. However, following the booster doses with at least 5 micrograms of antigen, glycosylated and deglycosylated proteins induced comparable antibody titres. There was no significant difference in the ability of the glycosylated or deglycosylated proteins to protect either the upper or lower respiratory tracts of immunized hamsters against PIV-3 challenge. These results suggest that the carbohydrate moieties of the HN and F proteins are not necessary for eliciting a protective response in hamsters.

Comparative analysis of the immunostimulatory properties of different adjuvants on the immunogenicity of a prototype parainfluenza virus type 3 subunit vaccine.

The immunogenicity of a parainfluenza virus type 3 (PIV-3) subunit vaccine consisting of affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in guinea-pigs and hamsters. The ability of several different immunopotentiating agents to enhance the antibody response of animals to the PIV-3 surface glycoproteins was evaluated. The immunity induced by HN and F alone was compared with the response elicited by purified proteins combined with Freund's complete adjuvant, aluminium phosphate, Syntex's threonyl-muramyl dipeptide (MDP) SAF-MF formulation, or Ribi's adjuvant formulation containing BCG cell wall skeleton (CWS), trehalose dimycolate (TDM) and monophosphoryl lipid A (MPL) in a 2% squalene-in-water emulsion. Purified proteins were also incorporated into three different liposome formulations prepared by the detergent dialysis procedure. Immunization of guinea-pigs and hamsters with two 15 micrograms doses of the PIV-3 surface glycoproteins administered in the absence of adjuvant elicited high haemagglutination inhibition, neutralization and anti-fusion titres. The liposome preparations failed to enhance the antibody titres. Ribi's adjuvant formulation was effective at inducing a good secondary response to the purified proteins while the immunostimulatory effects of aluminium phosphate, Syntex and Freund's adjuvants were clearly demonstrated in both primary and secondary responses. When administered without adjuvant, a 15 microgram dose of the HN and F mixture was capable of protecting hamsters against live virus challenge. The immunoprotective dose of the purified proteins could be reduced to at least 0.1 microgram by the addition of aluminium phosphate, Syntex or Freund's adjuvants.

Evaluation of the immunogenicity and protective efficacy of a candidate parainfluenza virus type 3 subunit vaccine in cotton rats.

A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.

Inhibition of subtilisin by influenza virus infected allantoic fluids.

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.

Allantoic fluid protease activity during influenza virus infection.

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.

Effects of influenza virus replication on neutral proteolytic activities in embryonated egg fluids.

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.

Protease susceptibility of human A influenza virus polypeptides.

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.