7.2.1. A highly configurable ELISA for the subtype-specific detection and characterization of anti-influenza virus antibodies will be available
Independent of the test format (indirect or blocking) and the type of antigens being employed, the ELISA will be available in a highly flexible and configurable format, allowing a tailor-made setup to be used depending on the intended use: (i) In its simplest format, the ELISA will only contain one HA subtype, and, if simultaneous NA subtyping is required, one NA subtype. In this format replicates of three columns of a 96-well plate will be coated with negative antigen (or left blank, if HA and NA antigens are not used as fusion proteins), HA and NA antigens, respectively. This format would be suitable for screening purposes, allowing the simultaneous testing of 28 (single wells) or 14 (duplicate wells) sera per plate. Based on the current worldwide spread of H5N1 highly desirable uses of this test format would e.g. be the differentiation of H5N1-specific antibodies from H5Nx and HxN1 antibodies, or, even simpler, just the screening for H5 subtype-specific antibodies. In this case, a maximum number of 92 sera could be tested on one plate, if single wells and a blocking ELISA format are used (which does not require the inclusion of negative antigen wells).
A second major application would be the use of the ELISA in the context of a DIVA vaccine: As it is currently practiced for zoo birds, these animals are vaccinated with an H5N2 vaccine in order to protect them from H5N1 infection. Here, the ELISA can be used as a bi-specific test by using H5 HA as well as N1 and N2 NA. The discrimination between H5N1-infected, H5N2-vaccinated, and unvaccinated/uninfected/infected with heterologous subtypes (non-H5/H7, non-N1/N2) birds would then be possible by looking at the H5-specific reactivity and the NA specificity in all those cases where H5 antibodies are detected: Sera from uninfected/unvaccinated birds will not show any H5-specific antibodies (unless they were previously infected with an H5 LPAIV), whereas vaccinated birds will be identified based on their positive H5 and N2 reactivity. H5N1-infected birds on the other hand will be identified based on their positive reactivity with the N1 antigen (irrespective whether they had also been vaccinated with H5N2 vaccine). Another use of the DIVA principle would be the vaccination of poultry with an inactivated vaccine containing the homologous HA but a heterologous NA, a strategy which has been used successfully in Italy to eradicate cases of H5 LPAIV infections (Capua et al., 2002).
At the other end of the available test formats it would also bee possible to set up the ELISA as an array for universal HA or/and NA subtype characterization. This would be done by including all HA subtypes of interest on one ELISA plate (in separate wells). The most feasable format for universal HA typing would be to use a blocking-ELISA format (no mock antigen wells needed), with replicates of H1 through H16 HA coated into 16 consecutive wells. This would allow to type 5 sera per plate (one series of 16 wells reserved for control reactions with 16 HA reference sera). To this end, the most sophisticated array format would be the combination of the 16 HA with the 9 NA antigens in a two-dimensional array format, where all the different combinations of HA with NA antigen would be coated by mixing one HA and one NA, respectively, in one well of on the plate. However, such a format would require the use of 384-well plates in order to include all the necessary controls. Furthermore, such an array format would require a considerable amount of test serum (unless the sera can be used sufficiently diluted), which is not feasable for sera from small bird species.
The use of highly influenza virus subtype-specific antigens, eventually in combination with subtype-specific polyclonal antisera or mabs will result in the availability of a highly customizable, highly configurable ELISA, depending on the questions to be addressed. The ELISA will be available in various formats (indirect – single serum dilution; indirect – multifold serum dilution; blocking/competition using either unlabeled or PO-labeled indicator antibodies.
Taken together, a test system for influenza virus laboratory diagnosis will be available for a broad range of very different types of studies to be performed, such as (i) rapid typing of antibodies, (ii) use of the ELISA as a DIVA diagnostic tool, (iii) epidemiological tracing, (iv) for surveillance projects e.g. aiming at determining the global distribution of selected influenza virus subtypes, (v) the present test system can be used both in veterinary as well as human medicine.
7.5.2. The materials obtained in the present project can be used as reagents for additional purposes
The HA and NA subtype-specific antigens and antibodies (polyclonal sera, mabs) produced in the present work will not only serve as tools in an antibody ELISA, but will also be available for several additional purposes. However, these additional applications are beyond the time frame and personal capacity of this project and would require additional manpower, or they could be used as starting materials in future projects:
(i) A new generation of antigen ELISA could be developed in the form of a sandwich ELISA, where e.g. polyclonal antisera (most likely anti-HA antisera) are coated onto the ELISA plate and would function as catching antibody to trap the homologous influenza virus subtype. The immobilized influenza virus would then be detected by a (PO-labeled or unlabeled) mab being specific for the respective homologous subtype.
(ii) Other test formats, such as lateral flow strip test, could be developed both for influenza virus subtype-specific antigen and antibody detection. The availability of a complete set of recombinant specific antigens and mabs against all (relevant) subtypes would allow to very quickly develop such tests for any given influenza virus subtype. This could be important if new influenza virus strains are detected for which immediate typing is required, e.g. if a new virus strain arises, bearing the potential to cause an influenza pandemic.
(iii) Both the recombinant antigens and the mabs represent promising tools to be applied in novel diagnostic techniques. For instance, these reagents could be used for subtype-sprecific high-throughput testing of swab samples (for virus detection) or screening of sera (for antibody detection) using fluorescence polarization technique (Jolley and Nasir, 2003). A second appealing application is the use of these reagents in biosensor technology, where either antigens or antibodies are coupled to nanosensors, allowing an extremely sensitive detection of either pathogens or antibodies. Among many available techniques, cantilever sensors will be considered as the most promising technology, since this technique has already been described for the detection of microbial pathogens (for review, see Hansen and Thundat, 2005).
7.5.3. No special biosafety requirements are necessary for production and use of this test system
The HA and NA typing of antibodies (or at least the production of the necessary reagents, i.e. the reference virus strains) requires the use of infectious virus when traditional typing methods such as the HI or SNT are employed. Such work can only be carried out under increased biosafety levels (at least BSL2 for non-H5, non-H7 viruses, at least BSL3 for H5N1 HPAIV). In contrast, the production of the reagents described here do not require special biosafety measures related to the protection of the personnel or the environment against influenza virus, since all the materials used in the ELISA are non-infectious. No matter what ELISA format is being used to detect antibodies, the test can be performed in any diagnostic laboratory. The absence of any special biosafety requirements is also a significant advantage in the context of any collaboration of our laboratory with other laboratories in the frame of this project (Veterinary Virology, University Zurich, see below), or in a later phase for any potential technology transfer to a commercial ELISA manufacturer.
7.5.4. Many new fundamental data about the characteristics of HA and NA epitopes will be obtained.
Although the primary purpose of this project is the development of universally applicable diagnostic reagents for influenza virus serology, a significant amount of new data will be obtained during the development and characterization of the reagents. These include:
(i) Mapping and characterization (linear vs. discontinous) of influenza virus subtype-specific epitopes on the HA and the NA protein
(ii) Information about the cross-reactivity of HAs and NAs of different subtypes: which epitopes are subtype-specific but conserved within one subtype (these are the ones to be used as diagnostic tools), which are conserved among several/all subtypes, and which epitopes are strain-specific and not conserved at all?
(iii) Correlation of genotyping and phenotyping based on antigen reactivity: which HA and NA gene fragments are most suitable for subtyping influenza viruses? What is the minimal length of sequence to be analyzed?
(iv) Role of certain (subtype-specific?) epitopes in the pathogenesis of influenzavirus infections (host specificity, cell/organ tropism, pathogenicity)
