Effect of lipid modification on the physicochemical, structural, antigenic and immunoprotective properties of Haemophilus influenzae outer membrane protein P6.

The outer membrane lipoprotein, P6 of Haemophilus influenzae was studied to determine the importance of the native palmitoyl moiety on its physicochemical and immunological properties. A recombinant P6 (rP6) molecule devoid of lipidation signal sequence was expressed in Escherichia coli and its properties were compared to those of the palmitylated protein purified from H. influenzae. The isoelectric point of rP6 was more acidic than that of the native protein and also exhibited less secondary structure than P6 as judged by circular dichroism. However, both forms of P6 induced identical P6-specific antibody titers in guinea pigs when Freund's adjuvant was used. These antisera reacted with a panel of overlapping P6 peptides in a comparable manner and in addition, rabbit antisera raised against the P6 peptides reacted equally well with P6 and rP6. Furthermore, all human convalescent sera tested exhibited similar anti-P6 and anti-rP6 antibody titers. However, rP6 was less immunogenic than P6 when administered either without adjuvant or in alum and when tested in competitive inhibition studies with anti-P6 antibodies, was a less effective inhibitor than native P6, suggesting a diminution in some of the antigenic activity of rP6. In spite of these differences, rP6 was capable of eliciting a protective antibody response against live H. influenzae type b challenge in a modified infant rat model of bacteremia. These findings demonstrate that the non-fatty acylated rP6 could possibily be substituted for native P6 in a vaccine against H. influenzae.

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.

Hybrid genes over-express pertactin from Bordetella pertussis.

Pertactin is a surface adhesin of Bordetella pertussis which is produced in small quantities when expressed from the native prn promoter. Hybrid genes were constructed in which the prn promoter was replaced by either the fha or tox promoter. Recombinant B. pertussis strains containing chromosomally integrated hybrid tox promoter/prn (toxpprn) or fha promoter/prn (fhapprn) genes expressed pertactin at approximately 5- and 8-fold the wild-type level, respectively. The pertactin was correctly processed and secreted and was biochemically and antigenically comparable to its wild-type counterpart, as determined by N-terminal sequence analysis, immunoblotting, peptide mapping, circular dichroism and antigenicity studies. In an adherence assay, a strain over-expressing pertactin was no more adherent than the wild-type strain, but a pertactin-deficient strain was less adherent.

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.