J Immunol Methods. 2015 May;420:18-23.

Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating infection from vaccination.

Bruderer U1, van de Velde J2, Frantzen I3, De Bortoli F4.
  • 1Discovery & Technology Vaccine Analysis, MSD Animal Health, Boxmeer, The Netherlands. Electronic address: urs.bruderer@merck.com.
  • 2Discovery & Technology Vaccine Analysis, MSD Animal Health, Boxmeer, The Netherlands.
  • 3Discovery & Technology Expression, MSD Animal Health, Boxmeer, The Netherlands.
  • 4Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany.



Serological differentiation between infection and vaccination depends on the detection of pathogen specific antibodies for an epitope that is modified or lacking in a vaccine. Here we describe a new assay principle that is based on differences in the binding properties of epitope specific antibodies. C-DIVA is a potent Classical swine fever vaccine candidate that differs from the parental C-strain life attenuated vaccine in the highly immunogenic TAVSPTTLR epitope by the deletion of two and the mutation of one amino acid (TAGSΔΔTLR). We show that C-DIVA vaccination elicits antibodies with high affinity for both the TAGSΔΔTLR and TAVSPTTLR epitope, whereas infection elicits only TAVSPTTLR specific antibodies. Differentiation is achieved with a double competition assay with negative selection for antibodies with affinity for the TAGSΔΔTLR epitope followed by positive selection for antibodies with affinity for the TAVSPTTLR epitope. Our findings add a new strategy for the development of marker vaccines and their accompanying discrimination assays and offer an alternative to the devastating stamping out policy for Classical swine fever.

KEYWORDS: Classical swine fever; Discrimination assay; Immune response; Marker vaccine

PMID: 25825375



We introduce a new method of distinguishing between epitope specific polyclonal antibodies which is based on binding properties. The practical relevance of this method is demonstrated by the generation of a new screening assay that finally converts a marker vaccine candidate for the devastating classical swine fever into a potent marker vaccine. The resultant ability to distinguish between infected and vaccinated animals will avoid the tragic and economically catastrophic killing of millions of pigs, infected or otherwise, until now the only method of dealing with classical swine fever outbreaks in Europe (1-3).

Apart from this practical aspect our findings also provide new insight into basic immunological mechanisms about the generation and regulation of antibody specificity and affinity. Our findings are based on the observation that the modification or even the elimination of immunodominant epitopes is not sufficient for the discrimination between vaccinated and infected animals (4-6). We identified a potential marker vaccine of which the modification of the most immunogenic epitope did not result in a lack of epitope specific antibodies but rather in a shift of their binding properties. Based on the uniformly high affinity of vaccine induced antibodies for the modified epitope that antibodies in infected animals completely lack, the detection of vaccine induced antibodies can be abolished through the use of a filter containing the modified epitope.

In addition to its relevance for the eradication of classical swine fever our findings can also be used as a template for further similar applications, providing a novel strategy for the development of new marker vaccines and their accompanying discrimination assays.



Fig. 1. Differentiation between classical swine fever infection and vaccination. Differentiation is based on an epitope specific asymmetry in the immune responses between infected and vaccinated pigs. Infected pigs readily elicit monovalent antibodies against the immunodominant TAVSPTTLR epitope but only vaccinated animals elicit bivalent antibodies with high affinity for both the TAVSPTTLR epitope and TAGSΔΔTLR epitope of the marker vaccine. ELISA based serological discrimination is achieved by preventing the binding of bivalent TAGSΔΔTLR specific antibodies to the solid phase by an excess of E2 expressing TAGSΔΔTLR in the liquid phase. The monovalent TAVSPTTLR specific antibodies are detected due to their capacity to prevent the binding of TAVSPTTLR specific monoclonal antibodies.


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