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Updated: May 29, 2026
Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses
Published on: April 4, 2019
Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus
Luke O'Shaughnessy1, Michael Carr, Brendan Crowley
1National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland. luke.j.oshaughnessy@nuim.ie
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.
Insights
This study developed new diagnostic tools for Human metapneumovirus (HMPV) by creating recombinant antigens and ELISA tests. Findings reveal high HMPV IgG seroprevalence and demonstrate HMPV-specific B cell memory, aiding future vaccine development.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Human metapneumovirus (HMPV) causes significant respiratory illness and asthma, with high global infectivity.
- Immunocompromised individuals are highly susceptible to HMPV infection.
- Lack of available HMPV vaccines and diagnostic reagents hinders effective management.
Purpose of the Study:
- To produce a range of HMPV recombinant antigens.
- To establish standardized ELISA methods for detecting HMPV IgG antibodies.
- To investigate HMPV-specific B cell memory for future vaccine research.
Main Methods:
- HMPV RNA isolation and RT-PCR.
- Cloning and expression of HMPV M and P genes in E. coli.
- Cloning and expression of HMPV N gene in insect cells via baculovirus system.
- Purification of recombinant antigens and development of HMPV-specific ELISAs.
Main Results:
- High-yield expression and purification of HMPV M and P antigens in E. coli.
- Successful expression and purification of recombinant HMPV N antigen.
- 99% HMPV IgG seroprevalence detected using M, N, and P antigens.
- HMPV M antigen showed highest diagnostic utility (99% reactivity).
- Demonstration of HMPV-specific B cell memory in 40% of tested individuals.
Conclusions:
- Development of robust diagnostic systems for HMPV detection.
- New insights into HMPV-specific B cell memory established.
- Foundation laid for future HMPV vaccine development and therapeutic strategies.
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