Improving heat stability of haemagglutinating antigens for avian influenza
Introduction
Avian influenza (AI) diagnostic reference reagents are used worldwide for influenza surveillance, providing the essential components for the application of standardised diagnostic protocols in samples obtained from animals and humans. International reference laboratories ship significant quantities of reagents globally as part of their mandate. It is in fact essential that reagents are produced with reference strains and according to standard procedures, so that quality standards are maintained. The haemagglutination inhibition (HI) test is widely used because it yields quantitative subtype-specific results for the serological diagnosis of influenza infections [1], [2]. In general 4-8 haemagglutinating units are used in the serological assay. It is therefore desirable to have stable reagents that do not undergo a decrease in titre following reconstitution or storage under optimal or sub-optimal conditions. In particular dispatch to remote laboratories may result in delays in the forwarding of product due to the unavailability of adequate paperwork, local public holidays or lengthy customs clearance. This often results in interruptions of the cold chain and thus deterioration of product.
The transport and storage conditions of these reagents are extremely important in order to preserve their antigenic properties and performance. One of the keystones in the improvement of reagents stability is the dry state achieved through the freeze-drying process (lyophilisation). However, extended exposure to sub-optimal temperatures may cause deterioration of the antigen even in its freeze-dried state, resulting in reduction or loss of haemagglutinating activity [3].
Carbohydrates have been reported to have lyoprotectant activity retaining the antigenic and immunogenic properties of biological reagents. Sucrose and D-(+)-Trehalose are two sugars which have been widely used as preservatives particularly in vaccine formulations [4], [5], [6].
D-(+)-Trehalose is a natural alpha-linked disaccharide, known to protect biomolecules, that can be found in animals, plants, and microorganisms. This lyoprotectant has been shown to be effective in stabilizing lipid membranes as well as proteins upon freeze-drying [7], [8], [9]. The stabilizing effect of Trehalose has been explained by the formation of a matrix which strongly reduces diffusion and molecular mobility (vitrification) and acts as a physical barrier between particles or molecules. Both the lack of mobility [10], [11] and the physical barrier [12], [13] provided by the glass matrix, prevent aggregation and degradation of the dried material, preserving its characteristics [14]. Moreover Trehalose is totally non-reducing, non-reactive and non-toxic, making it particularly suited as a lyoprotectant for reagent production and vaccine formulation [15], [16].
In order to improve the stability of our diagnostic reagents, particularly in relation to the effect of inadequate storage temperatures, we assessed the effect of the addition of D-(+)-Trehalose to our influenza antigens following a 2 week incubation at 37 °C or at 45 °C in order to mimic the duration of a delayed shipment in tropical or subtropical countries. In addition we evaluated the effect of D-(+)-Trehalose on long term storage of the reagents.
Section snippets
Antigen production
Three AI viruses (H7N3 A/Turkey/Italy/9289/V02; H5N2 A/Turkey/Italy/80; H9N7 A/Turkey/Scotland/1/70) were selected and propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs, according to EU Council Directive 2005/94/EC [1]. Allantoic fluid containing viruses was harvested, clarified and filtered, then inactivated using β-propiolactone. Briefly, β-propiolactone was added to the filtered allantoic fluid at a concentration of 0.2%. The solution was incubated under stirring
Results and discussion
Before initiating the study, each antigen was tested to determine whether the presence of D-(+)-Trehalose had an inhibitory effect on its HA and HI activity. The inactivated antigens, treated with and without D-(+)-Trehalose, were tested by HA assay in order to calculate the 4 Haemagglutinating Unit (4 HAU) that were subsequently used in an HI assay against positive and negative reference sera. No inhibitory effect on the HA and HI activity of the antigens was observed (data not shown).
To
Acknowledgements
This study was funded by the EU project FLUTRAIN “Training and technology transfer of Avian influenza diagnostics and disease management skills”.
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2012, VirologyCitation Excerpt :Increasing amounts of data are available on H5N1 HPAI infectivity under chemical and physical conditions (Lenes et al., 2010; Paek et al., 2010; Rice et al., 2007; Shahid et al., 2009; Wanaratana et al., 2010). The majority of studies investigate the effect of disinfectants on viral infectivity or the infectivity of HPAI viruses in water (Brown et al., 2009; Stallknecht et al., 2010) but few address the direct effect of chemical and physical factors on influenza virus infectivity (Dundon et al., 2007; Pizzuto et al., 2011; Terregino et al., 2009). The widespread infection of farm animals with HPAI H5N1 throughout Asia, Africa and Europe has raised concerns about the safety of animal products for human consumption and for the risk of transmission of infection to animals through the feeding of swill.