Improving heat stability of haemagglutinating antigens for avian influenza

Abstract

The increasing demand for avian influenza diagnostic reagents worldwide, has included requests for significant supplies of product to developing countries. Difficulties in dispatching to remote areas and tropical countries are a major concern to suppliers, international organisations and donors as delays in forwarding parcels often result in storage at non-optimal or inadequate temperatures results in loss in titre and thus wastage of resources.

In this study we demonstrate that the heat stability of avian influenza haemagglutination inhibition antigens of the H5, H7 and H9 subtype following 14 days of exposure to 37 °C and 45 °C is significantly increased by adding D-(+)-Trehalose to the freshly prepared antigen. Increased stability was detected both for freeze-dried antigens over an extended period of 6 months and also in heat exposed antigens that were then stored at +4 C for up to 35 days post reconstitution.

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.