Determination of the virus type/subtype/serotype involved in field outbreaks has to be estab- lished before within laboratories to permit proper control/ vaccination programs to be followed. Various techniques have been used to diagnose viral disease and to ascertain the serotype of the virus. These techniques are used for identification and screening of relevant antigens which is the first stage of pre-clinical development of vaccines followed by creation of the vaccine concept i.e. the mode or type of vaccine that is used to deliver the protection (discussed in the previous section) in the next stage. Below we discuss three methods that are used for identifica- tion of antibodies in FMDV (Virus Neutralisation Test), influenza (Hemagglutination Inhibition Test) and Ebola (Enzyme Linked ImmunoSorbant Assay) since they will be referred to in the following chapters.
2.2.2.1 Virus Neutralisation Test (VNT)
Neutralisation of a virus is defined as the loss of infectivity through reaction of the virus with specific antibody. Virus and serum are mixed under appropriate conditions and then inocu- lated into cell culture, eggs or animals. The presence of unneutralised virus may be detected by reactions such as cytopathic effect (CPE6),haemadsorption/haemagglutination (refer to Sec- tion 2.2.2.2 for details), plaque formation or disease in animals. The VNT is carried out as a two dimensional checkerboard titration in microtitre plates as described previously by Booth et al.[42]. This test uses an antiserum raised against a vaccine strain. The titres of this serum against a 50% tissue culture infective dose (100 TCID50) of the homologous vaccine strain and the same dose of a field isolate are compared to determine how antigenically ‘similar’ the field virus is to the vaccine strain, where similarity leads to neutralisation.
The procedure is as follows:
1. The viral field isolates are grown on cell cultures until adapted to give 100% CPE in 24 hours. Once adapted, the virus titre (log10 TCID50/ml) is determined by end-point titration.
2. A chequerboard titration [358] of virus against vaccine serum and along with a back- titration of virus alone is performed. Cells are added and incubated at 37◦C for 2-3 days after which they are assessed for CPE.
3. At each virus dose tested the corresponding antibody titres of the vaccine serum against the vaccine strain is determined by the method of Spearman-Karber [238]. Regression is then used to estimate the titre of the vaccine serum against each virus. The relationship between the field isolate and the vaccine strain is then expressed as an r1 value as:
r1 =
reciprocal arithmetic titre of reference serum against field virus reciprocal arithmetic titre of reference serum against vaccine virus
The reference serum in the above equation is a standard bovine serum (for details refer to the OIE “Manual of standards for diagnostic tests & vaccines” [94]). An r1 value is accepted only after achieving at least two consistent results.
4. Interpretation of the results: r1 values between 0.4 and 1.0 are indicative of reasonable levels of cross protection, whilst values below 0.2 indicate the need for a new vaccine for the isolated field virus [305]. A value of 0.3 has also been proposed as a conservative threshold [19] indicating the need for a new vaccine for a value below that.
5. The number of times that the examination is repeated [299] decides the confidence with which r1 values can be taken to indicate the antigenic relationship between strains. And thus these tests are repeated at least three times.
2.2.2.2 Hemagglutination Inhibition Test (HIT)
The nucleic acids of various viruses encode surface proteins that agglutinate the red blood cells (RBC) of a variety of species. For example; influenza virus particles have an envelope protein called the hemagglutinin, or HA, which binds to erythrocytes , causing the formation of a lattice. This property is called hemagglutination. Hemagglutination (HAI) assay apply the process of hemagglutination reaction of viral hemagglutinins with the sialic acid receptors on the surface of RBCs results in a lattice of agglutinated cells which settle irregularly in a tube or microtiter well. Unagglutinated cells settle in a compact button.
The basis of the HAI assay is that antibodies to that particular virus (for example-influenza virus) will prevent attachment of the virus to RBC. Therefore hemagglutination is inhibited when antibodies are present.
The procedure is as follows [376]:
1. RBCs from an appropriate species (Chicken, goose, guinea pig, etc.) is collected in Al- sever’s solution or heparin solution.
2. Diluent (e.g. bovine albumin veronal buffer) at appropriate pH, solutions to remove non- specific hemagglutinins from serum and infected cultural fluid or standard antigen (e.g preparation of influenza virus) for serology are also needed.
3. A preparation of virus (e.g. influenza viruses) with known HA titer or its HA titer is obtained.
4. Two-fold dilutions (e.g. from 1:4 to 1:1024) of patient/test serum are prepared for testing. 5. A fixed amount of virus is added to every well of a 96-well plate, equivalent to 4 HA units
(varies according to virus), except for the serum control wells.
6. The plate is then allowed to stand at room temperature for 60 minutes (time varies ac- cording to specific requirements).
7. RBCs are added and incubated at 40◦C for 30 minutes.
8. The wells are then read (for details of how the wells are read, refer to Grimes [128]). The highest dilution of serum antibody that prevents hemagglutination is called the HAI titer of the serum. A smooth or jagged shield of cells or an irregular button indicates agglutination.
9. If the serum contains no antibodies that react with virus, then hemagglutination will be observed in all wells. Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted.
2.2.2.3 Enzyme Linked ImmunoSorbant Assay (ELISA for Ebola)
The purpose of an ELISA is to determine if a particular antibody (or protein) is present in a sample and if so, how much. ELISAs are performed in 96-well plates which permits high throughput results. To obtain the elisa values for the Ebola dataset used this study, the following method was used [337]:
1. The bottom of each well is coated with one of the six structural proteins to which the primary antibody (HLA-A2) to be measured will bind.
2. TAP-deficient T2 cells expressing HLA-A2 were supplemented with 20% fetal bovine serum.
3. The serum is incubated in wells, and each well contains a different serum. A positive control serum and a negative control serum were included among all the samples being tested.
4. After some time, the serum is removed and weakly adherent antibodies are washed off with a series of phosphate buffered saline rinses.
5. To detect the bound antibodies, a secondary antibody (HLA-A2.1) was added to each well. The secondary antibody binds to the primary antibodies and and in this case was produced in a six to eight week old transgenic mice. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into coloured products.
6. After incubating the wells overnight at 37 degree centigrade, the secondary antibody so- lution is removed and loosely adherent ones are washed off with serum-free medium and then incubated with individual peptides at 37 degree centigrade for 18 hours.
7. The final step is the addition the enzyme substrate and the production of coloured product in wells with secondary antibodies bound.
8. When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density (i.e. the amount of coloured product) is determined for each well. The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells.
9. The HLA-A2.1 epitope comprising of the peptide GILGFVFTL derived from influenza matrix antigen, was used as a positive control [234] and the HLA-A2.1 nonbinder com- prising of the peptide IAGNSAYEY was used as a negative control [37].
2.3
Selection of Vaccines
Conventional methods of vaccine selection are time consuming and have failed to provide a solution for many pathogens. Availability of genome sequencing data has enabled the use of genetic information for vaccine development in silico.