The immunogenic epitopes of the glycoproteins of human metapneumovirus

Abstract

Recently discovered in 2001, human metapneumovirus (HMPV) is a member of the Paramyxoviridae family and is a major cause of respiratory tract infections in infants and young children as well as the elderly and immunocomprimised. In the related pneumovirus RSV, the two major surface glycoproteins, F and G are protective antigens in animal models although F is highly conserved and G highly variable. In this study, the equivalent glycoproteins of HMPV, F and G, were cloned into vaccinia virus to allow expression of the individual proteins. These recombinants were utilised for the generation of both monoclonal and glycoprotein specific polyclonal antibodies. Immunofluorescence studies revealed that the anti-F protein specific antibodies were cross reactive between both sub-groups and that anti-G antibodies were to a lesser extent, also cross reactive. These antibodies were also shown to neutralise homologous virus. Whilst anti-F protein antibodies also neutralised a heterologous strain of HMPV, so did anti-G antibodies directed towards a sub-group B but not a sub-group A strain. Western blotting with F and G glycoprotein specific anti-sera was unsuccessful due to high levels of non-specific reactivity in the sera. The generation of monoclonal antibodies towards the G glycoprotein was attempted by means of a novel screening system using inactivated recombinant vaccinia virus. However, due to the non-specific reactivity of the hybridomas with vaccinia virus, only one anti-G antibody was isolated along with two anti-F MAbs and one antibody directed towards an internal HMPV protein. Further characterisation of this antibody, by western blotting, indicated it was directed towards the phosphoprotein. The third surface glycoprotein of HMPV, SH, is larger than the equivalent in RSV and unlike the latter, may also play a role in protective immunity. Attempts to clone the HMPV SH gene identified several mutations in the sequence resulting in truncation of all or the majority of the lumenal domain of the proteins arising on adaption to replication in cell culture. SH glycoprotein specific antibodies generated against the recombinant vaccinia virus expressing the mutated SH protein of the B1 v strain of virus were cross reactive with an A2 strain in immunofluorescence studies and also neutralised wild type HMPV. The tendency of the virus to mutate on adaptation to replication in cell culture frustrated attempts to establish an animal model. In mice, whilst low passage virus, with a mixed population of wild type and mutant virus, resulted in a productive infection, high passage virus, with no functional SH glycoprotein produced an abortive infection with evidence of genome replication and transcription but no release of infectious virus. However, in challenge studies with mutant viruses mice immunised with HMPV were protected when challenged with the homologous strain, however, immunisation of mice with recombinant vaccinia virus expressing the G glycoprotein showed no signs of protection against challenge suggesting that in this animal model, the G glycoprotein is not a protective antigen.