O'Shaughnessy, Luke and Carr, Michael and Crowley, Brendan and Carberry, Stephen and Doyle, Sean (2011) Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus. Journal of Clinical Virology, 52 (3). pp. 236-243. ISSN 1386-6532

Preview

Download (958kB) | Preview

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. Study design: 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. Conclusion: This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

Item Type:	Article
Keywords:	Human metapneumovirus; HMPV; Baculovirus expression; HMPV recombinant antigens; ELISA and B cell ELISpot assay;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	7393
Identification Number:	https://doi.org/10.1016/j.jcv.2011.07.018
Depositing User:	Dr. Sean Doyle
Date Deposited:	26 Aug 2016 14:47
Journal or Publication Title:	Journal of Clinical Virology
Publisher:	Elsevier
Refereed:	Yes
Funders:	Higher Education Authority (HEA), Health Research Board (HRB)
URI:	http://www.sciencedirect.com
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

Repository Staff Only(login required)

Item control page

Downloads

Origin of downloads