3R-Project 28-92

Analysis of the applicability of in vitro immunological methodologies to the study of foot-and-mouth disease vaccine efficacy

McCullough K. Ch.
Institut für Viruskrankheiten und Immunprophylaxe, IVI, Mittelhäusern

Duration: 4 years End of the Project: 1996

Background and Aim
The humoral response in sera against viruses is a well studied aspect of the immune response. A good example of these studies is that on foot and mouth disease virus (FMDV). Traditionally, the detection of specific antibody via titration and subsequent dilution of the serum antibody to eliminate background problems does not take into account the in vivo environment of the serum antibodies. It is therefore unclear how the characteristics of such reactions in a diluent buffer can be related to those in natural body fluid extracts such as serum. The reactivity of serum antibody, as an entity, is also influenced by the time of contact with the antigen; in most immunoassays, an incubation time of one hour is chosen.
Using vaccinated (FMDV serotype O1) and non-vaccinated cattle, we have demonstrated that the majority of the specific reaction between antibody and antigen in sera occurs within 10 to 60 seconds. In contrast, aspecific and non-specific reactions are still at low levels within such a short time span. When dilutions of sera were employed and compared with the original reactivity of the sera, it was observed that the use of diluted sera provided an assessment of antibody activity resulting from the dilutions, and not as pertaining directly to the reactivity in the serum itself. Results from serum which can be related to its capacity to function under in vivo conditions, can only be obtained when the in vitro analytical methodology creates conditions close to those found in the serum itself.

Method and Results
The objective was to identify in vitro means for measuring immunological potential in animals vaccinated against FMDV. Serum antibody avidity did not always relate to protection, whereas 10 and 60 second incubation times for serum with antigen identified protected animals. This test presumably permitted an analysis of combined reactive capacities for specific and aspecific (innate) serum components, but the reactivities were sensitive to freezing and thawing. It would be necessary to continue these analyses, to determine if serum samples should not be frozen/thawed, or if dilution with fresh non-immune serum would circumvent the problem.
The characteristics of an in vitro immunization system, as a means to replace animal immunization, was also investigated. Therein, the problem of culture-dependent programmed cell death (PCD) was identified, a solution for which was the provision of accessory cells and plasma - derived factors. These deliver the required signals to prevent PCD. Future work should concentrate in this area, in order to build an in vitro immunization system, wherein the viability and responsive capacity of the cells is more akin and relevant to that found in vivo.

References
1) Scicluna L.A. and Mc Cullough K.C. (1999) Rapidity of specific antibody-antigen interactions. Scand. J. Immunol. 50, 167-176.

2) Scicluna L.A., Bruckner L. and Mc Cullough K.C. (2001) Qualitative assessment of the humoral immune status against FMDV in post-vaccination cattle. Vaccine 19, 2975-2986.