title: Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus.
abstract: BACKGROUND
  Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens.
  
  
  OBJECTIVE
  To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies.
  
  
  METHODS
  HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA.
  
  
  RESULTS
  High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N- and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1-15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested.
  
  
  CONCLUSIONS
  This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.
authors.affiliation: National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland. luke.j.oshaughnessy@nuim.ie
authors.forename: Luke
authors.initials: L
authors.lastname: O'Shaughnessy
authors.forename: Michael
authors.initials: M
authors.lastname: Carr
authors.forename: Brendan
authors.initials: B
authors.lastname: Crowley
authors.forename: Stephen
authors.initials: S
authors.lastname: Carberry
authors.forename: Sean
authors.initials: S
authors.lastname: Doyle
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D016367:Baculoviridae / Q000235:genetics; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D007313:Insecta; D029121:Metapneumovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008875:Middle Aged; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018184:Paramyxoviridae Infections / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins