Michael W. Wathen

Functional analysis of human cytomegalovirus polymerase accessory protein.

The human cytomegalovirus (HCMV) UL44 gene product, polymerase accessory protein, was cloned and expressed in Escherichia coli as a 53,000 MW protein. The activity of HCMV DNA polymerase (Pol) alone and Pol/UL44 complex was evaluated in Pol assays designed specifically to elucidate Pol/UL44 interactions. Addition of UL44 to HCMV Pol with primed, single-stranded DNA resulted in increased incorporation of nucleotides into DNA, which was correlated with enhanced enzyme processivity. Several deletion mutants which span the UL44 sequence were constructed and examined for the ability to stimulate Pol activity and to bind double-stranded DNA. The functional domains of UL44 protein were determined to reside within the N-terminal 309 amino acids of the wild type sequence, since deletions within this region resulted in loss of DNA binding and the ability to stimulate Pol. Deletion of C-terminal amino acids 310-433 had no effect on the ability of UL44 protein to increase the processivity of HCMV DNA Pol.

Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit

Local IgA and IgG antibodies against respiratory syncytial virus (RSV) were induced in the respiratory tract of mice following intranasal vaccination with an RSV chimeric FG glycoprotein and cholera toxin B (CTB) as a mucosal adjuvant. Local antibody production was not induced following parenteral immunization with FG administered in alum adjuvant. While both vaccination protocols induced serum antibodies against RSV and protected the lower respiratory tract from RSV infection, only intranasal FG/CTB afforded protection of the upper respiratory tract. These data suggest that vaccination via the mucosal route may be superior to vaccination by a parental route in providing complete protection against RSV.

Characterization of a Novel Human Respiratory Syncytial Virus Chimeric FG Glycoprotein Expressed Using a Baculovirus Vector

Human respiratory syncytial virus (RSV) codes for two glycoproteins (F and G) which have been shown to the major targets for the host antibody response. We have expressed a novel chimeric glycoprotein (FG) in insect cells using a baculovirus vector. The chimeric glycoprotein contains the signal and extracellular regions of the RSV F glycoprotein linked to the extracellular region of the RSV G glycoprotein. Beginning at the amino terminus, the chimeric glycoprotein consists of amino acids 1 to 489 from RSV F followed by amino acids 97 to 279 from RSV G. The chimeric FG glycoprotein did not contain an anchor region and was efficiently secreted into the medium of recombinant baculovirus-infected insect cells. The FG glycoprotein ranged in size from 69K to 91K and was heterogeneous with respect to isoelectric point. The cleavage site present on the F glycoprotein was recognized on the chimeric FG, and the glycoprotein appeared to be antigenically similar to the native RSV F and G glycoproteins.

Protection of cotton rats against human parainfluenza virus type 3 by vaccination with a chimeric FHN subunit glycoprotein

A cotton rat model of experimental human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FHN, a novel chimeric glycoprotein which contains the extracellular regions of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of PIV-3. The FHN protein was expressed in insect cells using a baculovirus vector system. FHN vaccination resulted in induction of neutralizing antibodies, was completely protective at doses of 100 ng, and was superior to vaccination with secreted forms F and HN proteins, or mixtures of the F and HN glycoproteins. In addition, FHN immunization induced lymphoproliferative responses in mice which were directed against both the F and HN glycoproteins. Fusion of the F and HN proteins into a single chimeric glycoprotein appeared to enhance the protective immune response compared to that elicited by the individual glycoproteins or mixtures of the two glycoproteins.

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines

Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) cells. Comparison of the mammalian cell- and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cell-expressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoaffinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell- and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell- and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.

Development of a novel subunit vaccine that protects cotton rats against both human respiratory syncytial virus and human parainfluenza virus type 3

A cotton rat model of experimental human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FRHNP, a novel chimeric glycoprotein which contains the extracellular regions of the fusion glycoprotein of RSV and the attachment glycoprotein of PIV-3, as a single subunit vaccine against these two viruses. This work was prompted by previous cotton rat studies that demonstrated that the major protective antigens of the two viruses were these glycoproteins. FRHNP was expressed in insect cells using a recombinant baculovirus. Vaccination with FRHNP resulted in induction of both RSV and PIV-3 neutralizing antibody and doses of 200 ng completely protected rats from either RSV or PIV-3 challenge. These results demonstrate that in the cotton rat animal model a single chimeric glycoprotein can be an effective vaccine against both RSV and PIV-3.

A chimeric glycoprotein of human respiratory syncytial virus termed FG induces T-cell mediated immunity in mice

Popliteal lymph node cells taken from mice vaccinated with the FG glycoprotein were exposed in vitro to respiratory syncytial virus (RSV) antigens. Proliferation to FG or RSV antigens was blocked with anti-CD4 monoclonal antibody treatment. FG-vaccinated mice developed classical late delayed type hypersensitivity (DTH) reactions when exposed to FG antigen in vivo.

Vaccination with a heterologous respiratory syncytial virus chimeric FG glycoprotein demonstrates significant subgroup cross-reactivity

A subunit vaccine candidate, termed FG, is a chimeric glycoprotein composed of the extracellular domains of the fusion (F) glycoprotein and the attachment (G) glycoproteins of a subgroup A respiratory syncytial virus (RSV). Two subgroups, A and B, of RSV differ primarily within the G glycoprotein. Therefore, it has been suggested that a subunit vaccine composed of the G glycoprotein would need to contain the G glycoproteins from both RSV subgroups. We have engineered a second chimeric glycoprotein, FGB, which is composed of the F glycoprotein from RSV subgroup A and the G glycoprotein from RSV subgroup B and is expressed in baculovirus. A comparison of protection between the two subunit vaccines (FG and FGB) was performed in cotton rats after homologous and heterologous virus challenge. FG and FGB appeared to afford the same degree of protection against either homologous or heterologous challenge. Serum neutralization titres against homologous or heterologous virus were nearly equivalent following FG or FGB vaccination. Radioimmunoprecipitation using sera from rats immunized with FG or FGB revealed cross-reactivity between the two G glycoproteins. Adsorption of anti-F antibody from serum of rats immunized with FG significantly reduced the RSV neutralizing activity of the serum suggesting that enhanced neutralization previously observed with FG antisera compared with F antisera alone may not be entirely attributed to antibodies against the G glycoprotein but may be attributed to a function associated with the G glycoprotein portion of FG which enhances the immunogenicity of the F portion of FG.