JIM-11989; No of Pages 6 Journal of Immunological Methods xxx (2015) xxx–xxx

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Research paper

Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating infection from vaccination Urs Bruderer a,⁎, Jan van de Velde a,1, Inge Frantzen b, Francesca De Bortoli c a b c

Discovery & Technology Vaccine Analysis, MSD Animal Health, Boxmeer, The Netherlands Discovery & Technology Expression, MSD Animal Health, Boxmeer, The Netherlands Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany

a r t i c l e

i n f o

Article history: Received 16 February 2015 Received in revised form 18 March 2015 Accepted 18 March 2015 Available online xxxx Keywords: Classical swine fever Marker vaccine Discrimination assay Immune response

a b s t r a c t 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. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The inability to distinguish between infected and vaccinated individuals is an intrinsic limitation to the use of vaccines. This can be overcome by marker vaccines in combination with matching diagnostics that discriminate infection and vaccination by marker dependent differences in the immune response. However the immune response against added positive markers is not indicative of the quality of the vaccination. Alternatively Abbreviations: C-DIVA, China strain derived marker vaccine; CSF, Classical swine fever; DIVA, Differentiation between Infected and Vaccinated Animals; TAGSΔΔTLR, modified TAVSPTTLR epitope in C-DIVA; TAVSPTTLR, epitope of CSF E2. ⁎ Corresponding author. Tel.: +31 485 585 332; fax: +31 485 587 490. E-mail address: [email protected] (U. Bruderer). 1 Current address: Synthon Biopharmaceuticals, Microweg 22, Nijmegen, The Netherlands.

the creation of a negative marker of sufficient sensitivity requires the elimination or modification of a highly immunogenic epitope with the risk of reduced potency and viability of such vaccines (Dong and Chen, 2007; Eblé et al., 2013; Reimann et al., 2010). Here we describe a new marker principle applied to Classical swine fever (CSF). CSF is a highly contagious disease causing enormous financial losses and the mandatory preemptive slaughter of millions of pigs (Meuwissen et al., 1999; Paton and Greiser-Wilke, 2003; Stegeman et al., 2000). Live attenuated vaccines against CSF provide full protection within a few days after vaccination but have been no alternative to the stamping out policy because they do not fulfill the DIVA (Discrimination between Infected and Vaccinated Animals) criteria (Qiu et al., 2006; Schroeder et al., 2012). Only one CSF epitope, the linear TAVSPTTLR epitope meets the criteria of specificity and sensitivity as a target for serologic assays (Eblé et al., 2013; Schroeder et al., 2012). The TAVSPTTLR epitope is

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Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

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U. Bruderer et al. / Journal of Immunological Methods xxx (2015) xxx–xxx

unique because it is highly specific for CSF virus, highly conserved amongst all known CSF strains and highly immunogenic (Kortekaas et al., 2011; Lin et al., 2000; Liu et al., 2006). All vaccines with a modified or replaced TAVSPTTLR moiety still elicit antibodies interfering in the detection of TAVSPTTLR specific antibodies (Kortekaas et al., 2011; Reimann et al., 2010; Schroeder et al., 2012) which demonstrates the difficulty of discrimination on the basis of specificity for TAVSPTTLR. This dilemma demands either a further search for a new efficacious vaccine that does not elicit these interfering antibodies or a search for a new means of discrimination between TAVSPTTLR specific antibody populations elicited by infection and those elicited by an existing vaccine. Focusing on the latter we reexamined antibody populations elicited by an established live attenuated vaccine known as C-strain containing the wild type TAVSPTTLR epitope, and a derivative vaccine differing from the parental strain in the TAVSPTTLR epitope called C-DIVA. Cstrain mutants with progressively larger deletions of the TAVSPTTLR epitope resulted in highly reduced viability. CDIVA is the result of passaging C-strain mutants in persistently infected cells allowing the increase of their fitness by forced evolution (Kortekaas et al., 2011).

2.3. Assays IDEXX CSFV Ab Test Kit: Tests were performed according to the instructions of the manufacturer (IDEXX Livestock and Poultry Diagnostics, Liebefeld-Bern, Switzerland). Binding property assessment assay: The assay measures the affinity of polyclonal serum antibodies for either the TAVSPTTLR epitope of the recombinant C-strain E2 or the TAGSΔΔTLR epitope of the recombinant C-DIVA E2 (Fig. 1). It differs from the IDEXX CSF Ab Test kit only in that the sera are incubated in the presence of recombinant E2 in the liquid phase. This incubation step contains mixtures of 40 μl sample diluent, 20 μl serum, and 40 μl purified recombinant E2 molecules. Each serum was separately incubated in the presence of differing concentrations (600 μg/ml, 10 μg/ml, 3.33 μg/ml, 1.11 μg/ml, 0.37 μg/ml, 0.12 μg/ml, 0.041 μg/ml) of either Cstrain E2 and C-DIVA E2 diluted in PBS/BT (PBS containing BSA (0.1% w/v) and Tween 20 (0.05% v/v)) or PBS/BT as a control. In each plate a serial dilution of conjugate (100%, 50%, 25%, and 12.5% of the undiluted conjugate) was added in wells that had been incubated with PBS/BT during the first incubation step. The degree of inhibition due to the competition between

2. Materials and methods 2.1. Sera Previously described sera (Kortekaas et al., 2011) from vaccination/challenge experiments and sera from the same experiment, collected 70 days post vaccination, were provided by J. Kortekaas, (Virology Division, Central Veterinary Institute, part of Wageningen University Research Center, Lelystad, The Netherlands). Negative field sera from the Netherlands and sera from animals experimentally infected with several CSFV, BVDV, and BDV strains were provided by W.L. Loeffen, (Virology Division, Central Veterinary Institute, part of Wageningen University Research Center, Lelystad, The Netherlands). Sera from experimental infection of pigs included the CSF strains Alfort, Behring, Bergen, Brescia, Henken, Isol. Rusland, Melis, Paderborn, Spruit, and v. Zoelen. 2.2. Recombinant E2 molecules The E2 genes of vFlc34 (C-strain) and vFlc-ΔPTa1 (C-DIVA), codon optimized for expression from baculovirus vectors, were chemically synthesized and cloned behind the polyhedrin promoter in a pFastBac plasmid. The expression cassette was transferred by site-specific transposition in DH10Bac E. coli cells to a recombinant bacmid DNA in which chitinase and vcathepsin genes had been deleted. Bacmid DNA was used to transfect Sf9 insect cells. Recombinant baculovirus obtained from the transfection supernatant was used to infect fresh Sf9 insect cells. Subsequent virus stocks were used to infect Sf21 insect cells cultured in 2 L bioreactors. Both recombinant proteins were primarily expressed in the supernatant of these bioreactor cultures. Both recombinant E2 proteins were affinity purified using purified monoclonal antibody C2 (Kortekaas et al., 2010) coupled to Sepharose. E2 in supernatants was quantified by an ELISA measuring the inhibition of C2 binding to purified E2 on the solid phase by E2 in the liquid phase.

Fig. 1. Assessment of TAVSPTTLR specific antibody reactivities. Schematic diagram of TAVSPTTLR specific antigen antibody interactions. Constellation 1 depicts a standard CSF assay based competition between serum and monoclonal antibodies. Constellations 2 and 3 depict the reversal of the inhibition shown in Constellation 1 due to competition between E2 in the solid phase and in the liquid phase for binding to serum antibodies.

Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

U. Bruderer et al. / Journal of Immunological Methods xxx (2015) xxx–xxx

the E2 in the solid phase and in the liquid phase was calculated using the forecast function of Excel in the linear range of the conjugate titration between 50% and 12.5% binding corresponding to 50% and 87.5% inhibition. The C50 values, representing the concentration of competitor (recombinant C-strain E2 or C-DIVA E2) required to reverse 50% of the inhibition of conjugate binding to E2 by serum antibodies, were calculated according to Reed and Muench (Reed and Muench, 1938). Double competition screening assay: the test is a modification of the IDEXX CSFV Ab Test Kit. The procedure is the same with the exception that 50 μl sera are incubated in the presence of 25 μl 2× concentrated IDEXX sample diluent (kindly provided by C. Schelp, IDEXX Livestock and Poultry Diagnostics, Liebefeld-Bern, Switzerland) and 25 μl baculovirus cell culture supernatant containing 76 μg C-DIVA E2/ml. As for the IDEXX CSF Ab Test Kit the interpretation criteria were defined as negative for inhibition b30%, ambiguous for inhibition between 30% and 40%, and positive for inhibition N40%. 2.4. Modeling The crystal structure of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at neutral pH (PDB code 2yq2 (El Omari et al., 2013)) was modified to respectively carry the TAVSPTTLR epitope unique to CSFV and the mutated C-DIVA epitope TAGSΔΔTLR. These two new structures were then solvated using CHARMM (Brooks et al., 2009; El Omari et al., 2013) and after equilibration and heating an energy minimization was run. The resulting minimized structures were then aligned and compared using PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC). All distances were also measured using PyMOL. 3. Results 3.1. Assessment of TAVSPTTLR specific antibody populations As a tool for discrimination we produced recombinant E2 molecules by chemically synthesizing codon optimized genes of both the C-strain E2 and the C-DIVA E2. Recombinant E2 molecules were subsequently expressed in baculovirus infected insect cells and affinity purified. Affinity distributions of antigen specific antibody populations can be determined based on competition between antigen in the liquid and antigen in the solid phase for antibody binding (Bruderer et al., 1992b). Here we created a new assay principle for the assessment of epitope specific polyclonal antibody populations. The strategy for analyzing the affinity of TAVSPTTLR epitope specific antibodies for either of the two recombinant E2 molecules is shown in Fig. 1. Constellation 1 is the format of a standard E2 competition assay for the detection of CSF. Sera are incubated in wells with recombinant wild type E2 in the solid phase (A). Unbound serum components are removed in a washing step. Enzyme labeled monoclonal anti-TAVSPTTLR antibodies compete with the bound serum antibodies for binding to E2 in the solid phase (B). The degree of removed unbound monoclonal antibody after a second washing step (C) is a measure of the amount of TAVSPTTLR specific porcine serum antibodies. In Constellations 2 and 3 affinities of TAVSPTTLR specific porcine serum antibodies are measured in a double competition assay. In a first incubation step (A) recombinant

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wild type E2 in the solid phase competes with either C-strain E2 (Constellation 2) or C-DIVA E2 (Constellation 3) in the liquid phase for binding to E2 specific serum antibodies. Affinity distributions of TAVSPTTLR specific antibodies can be determined by a second incubation step (B). Serum antibodies bound to E2 in the solid phase compete with enzyme labeled monoclonal TAVSPTTLR specific antibodies for the binding to the TAVSPTTLR moiety of E2 in the solid phase. The concentration of the corresponding E2 molecules necessary to reverse inhibition of monoclonal antibody binding by 50% (C50) is used as a measure for the affinity of the serum antibodies for either the TAVSPTTLR (Constellation 2) or the corresponding TAGSΔΔTLR moiety (Constellation 3). A low C50 value is indicative of high affinity. The affinity distribution of sera 35 and 70 days post vaccination (dpv) with either C-strain or C-DIVA is summarized in Table 1. C-strain vaccination elicits antibodies that exhibit high affinity for the TAVSPTTLR moiety but do not recognize the modified TAGSΔΔTLR moiety (C50 N 600) of the recombinant CDIVA E2. Affinity maturation is indicated by a decrease in the C50 values from 35 dpv and 70 dpv. In contrast vaccination with CDIVA elicits antibodies with comparable affinities for both the TAVSPTTLR and the TAGSΔΔTLR moiety. Also the C50 values of these serum antibodies are lower 70 dpv as compared to 35 dpv. The difference in the affinities 35 dpv for the TAVSPTTLR moiety is statistically significant between antibodies immunized with Cstrain and C-DIVA (P 0.021 in an unpaired t test). Also the difference in the affinities of antibodies for the TAVSPTTLR and the TAGSΔΔTLR moiety is statistically significant 35 days after CDIVA vaccination (P = 0.035 in a paired t test). All groups exhibit a narrow range of average affinities. C-strain vaccination and C-DIVA vaccination elicited antibodies differing by more than a factor 1000 in their C50 values for the TAGSΔΔTLR moiety. These results demonstrate that C-DIVA vaccination elicits a TAVSPTTLR specific antibody population distinct from that elicited by infection or vaccination with the C-strain vaccine.

3.2. Evaluation of the double competition assay as a means for differentiation between infection and vaccination Having established these differences in the binding patterns we evaluated the potential of an assay format compatible with the needs of large scale monitoring in eradication programs of epidemic veterinary diseases. To this end the double competition format is no longer used for the assessment of affinity distributions but for filtering antibodies with affinity for the Table 1 Affinity distribution patterns of porcine anti-TAVSPTTLR serum antibodies upon C-strain and C-DIVA vaccination. Vaccination

C-strain

C-strain

C-DIVA

C-DIVA

Days post vaccination C50 (μg/ml) C-strain E2 C50 (μg/ml) C-DIVA E2

35

70

35

70

5.76 ± 2.9

0.53 ± 0.19

1.44 ± 0.49

0.09 ± 0.04

N600

N600

0.68 ± 0.04

0.06 ± 0.03

The affinity of porcine anti-TAVSPTTLR serum antibodies 35 or 70 days post vaccination (dpv) is expressed as the concentration (mean ± STD) of either Cstrain E2 or C-DIVA E2 molecules required to inhibit 50% of the binding of monoclonal anti-TAVSPTTLR antibody to the E2 on the solid phase.

Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

U. Bruderer et al. / Journal of Immunological Methods xxx (2015) xxx–xxx

3.3. Structural features of the TAVSPTTLR and the TAGSΔΔTLR epitopes

100 negative ambiguous

80

positive 60

40

20

Standard Assy

dp i 28

dp i 21

14 dp i

dp i

21

28

dp i

0 14 dp i

TAGSΔΔTLR moiety (Fig. 1, Constellation 3). The use of a standard assay confirms that both vaccinations with C-strain (Fig. 2A) and C-DIVA (Fig. 2C) elicit TAVSPTTLR specific antibodies. The sensitivity is not impaired by the double competition (Fig. 2B). In contrast the incubation of serum samples in the presence of an excess of recombinant C-DIVA E2 completely abrogates the interference of C-DIVA induced TAVSPTTLR specific antibodies (Fig. 2D). The specificity of the new screening assay was established by testing more than 400 negative field sera and sera from animals infected with the related Bovine viral diarrhea virus and Border disease virus. None of the tested negative field sera inhibited more than 11%, well below the 30% for ambiguous or positive samples. No cross-reactivity with sera exposed to Bovine viral diarrhea virus and Border disease virus was found. A comparison of the sensitivity of the commercial standard assay and the new double competition assay is shown in Fig. 3. Testing more than 200 sera from challenge experiments with 10 different CSFV strain showed that the percentage of positive sera 14 dpv was higher for the double competition assay (30%) as compared to the standard assay (14%). The difference diminishes over time (21 and 28 dpv).

sera (%)

4

Double Competition Assay

Fig. 3. Detection of CSF upon controlled infection of pigs by various CSF strains. The percentage of negative, ambiguous, and positive sera 14, 21, and 28 days post infection (dpi) is shown for a standard assay (IDEXX CSFV Ab Test Kit) and the Double Competition Assay.

BVDV E2 (El Omari et al., 2013) was used to compare the steric context of the TAVSPTTLR and TAGSΔΔTLR moieties within the CSF E2 molecules. The deletion of two amino acids in the CDIVA E2 results in a shortening of the epitope loop (Fig. 4B) which moves the relative position of the positive charge (Fig. 4C). 4. Discussion

The steric consequences of the modification of the TAVSPTTLR moiety in the C-DIVA E2 protein are shown in Fig. 4. Modeling based on X-ray crystallography data of purified

The concept of the C-DIVA vaccine is based on maintaining the established safety and efficacy of the parental C-strain virus

A

B 100

90

Pig 3160

90

Pig 3160

80

Pig 3159

80

Pig 3159

70

Pig 3158

60

Pig 3156

50 40 30 20

inhibition (%)

70 Pig 3158

60

Pig 3156

50 40 30 20

days post C-strain vaccination

56

49

42

35

days post C-strain vaccination

C

D

100

100

90

pig 3165

90

pig 3165

80

pig 3164

80

pig 3164

70

pig 3163

60 50

pig 3162

40

pig 3161

30 20

70

pig 3163

60 50

pig 3162

40

pig 3161

30 20

days post C-DIVA vaccination

56

49

42

35

28

21

14

56

49

42

35

28

21

-20 14

-20 7

-10

0

0

-10

7

10

0

0

10

inhibition (%)

inhibition (%)

28

21

0

56

49

42

35

28

-20 21

-20 14

-10

7

0

-10

0

0

14

10

10

7

inhibition (%)

100

days post C-DIVA vaccination

Fig. 2. Reactivity of individual porcine serum antibodies from animals vaccinated with C-strain (A, B) and C-DIVA (C, D) tested in a commercial standard E2 assay (Idexx CSFV Ab; A, C) and the newly developed double competition assay (B, D). In both assays inhibition values ≤30% are considered negative, between 30% and 40% suspect, and ≥ 40% positive for the presence of CSF specific antibodies.

Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

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Fig. 4. Steric configuration of C-strain and C-DIVA E2 epitopes. An overlay of the recombinant C-strain E2 (blue) and C-DIVA E2 (yellow) onto the structure of a closely related BVDV E2 is shown in (A). The TAVSPTTLR epitope is colored red. TAVSPTTLR and the TAGSΔΔTLR epitopes are shown in (B) and (C). The spatial distances (Å) between the corresponding amino acids are depicted for threonine (B) and the positively charged arginine (C) respectively. The modeling is based on the crystal structure of the BVDV1 E2 with the PDB code 2yq2.

combined with the new DIVA ability. The TAVSPTTLR epitope was modified in order to prevent positive results in CSF ELISAs based on competition between porcine serum antibodies and monoclonal antibodies for binding to the TAVSPTTLR epitope in recombinant E2 protein (Eblé et al., 2013; Schroeder et al., 2012). The occurrence of positive results in these assays demonstrates however that C-DIVA vaccination elicits antibodies that either have the capacity to recognize the TAVSPTTLR epitope or must bind to epitopes overlapping with the TAVSPTTLR moiety. Here we demonstrate that these epitope specific polyclonal serum antibodies can be discriminated according to their affinity for the TAGSΔΔTLR moiety of C-DIVA. We succeeded in the characterization of TAVSPTTLR specific serum antibodies with a double competition assay format utilizing recombinant E2 molecules expressing the TAVSPTTLR or the TAGSΔΔTLR moieties in the correct structural context. We show that TAVSPTTLR specific antibody populations resulting from infections are distinguishable from those elicited by C-DIVA. Both populations have the high affinity for TAVSPTTLR in common. In contrast C-DIVA vaccination results in antibodies with high affinity for TAGSΔΔTLR that is completely missing upon C-strain vaccination (Table 1). Despite the fact that animals vaccinated with C-DIVA had no exposure to the TAVSPTTLR moiety they exhibit a slightly higher affinity for this moiety than animals immunized with C-strain expressing the TAVSPTTLR moiety. This phenomenon where antibodies exhibit affinity for structures other than the immunogen is known as heteroclitic binding (Berzofsky and Schechter, 1981; Chitarra et al., 1993). Differences in the ability to induce a steric fit upon approximation (Bruderer et al., 1992a; Davies and Cohen, 1996; Sundberg and Mariuzza, 2002) may explain the ability of C-DIVA induced antibodies to bind to both TAGSΔΔTLR and TAVSPTTLR or the

lack of C-strain induced antibodies to interact with the shorter loop of the TAGSΔΔTLR moiety. Under physiological conditions specific binding has to overcome the repulsion between macromolecules (Van Oss, 1995). Antibody binding is described as a sequential process initiated by electrostatic interactions at the longest range followed by additional stabilizing interactions at a shorter range (Reverberi and Reverberi, 2007). The difference in the relative position of the positively charged arginine in the TAVSPTTLR and the TAGSΔΔTLR moiety (Fig. 3C) may explain the ability of TAVSPTTLR specific antibodies to recognize the TAGSΔΔTLR moiety. Differences in the ability to induce a steric fit upon approximation (Bruderer et al., 1992a; Davies and Cohen, 1996; Sundberg and Mariuzza, 2002) may explain the ability of C-DIVA induced antibodies to bind to both TAGSΔΔTLR and TAVSPTTLR or the lack of C-strain induced antibodies to interact with the shorter loop of the TAGSΔΔTLR moiety. Alternatively or additionally the TAGSΔΔTLR moiety may represent a stereochemical environment more permissive for cross-reactivity than that of the TAVSPTTLR moiety (Chitarra et al., 1993). The finding that the monoclonal anti-TAVSPTTLR antibodies of three commercial anti-CSF test kits exhibit no detectable affinity for the TAGSΔΔTLR moiety in the recombinant C-DIVA E2 (results not shown) suggest that the regions flanking the TAVSPTTLR epitope play a minor role. This view is consistent with our modeling experiments that revealed that the shortening of the TAVSPTTLR loop changes the relative position of the corresponding amino acids within the modified epitope but has relatively little influence on the flanking regions. Our results demonstrate that the differences in the binding patterns of TAVSPTTLR specific serum antibodies can be used to develop an assay format compatible with the needs of large

Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

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scale monitoring of porcine populations. Such an assay is based on a) the complete lack of antibodies with detectable affinity for the TAGSΔΔTLR moiety in the sera of infected animals and b) antibodies elicited by C-DIVA that exhibit comparable or higher affinity for the TAGSΔΔTLR moiety as compared to the TAVSPTTLR moiety. In this new screening assay the double competition format is no longer used for the assessment of affinity distributions but prevents antibodies with affinity for the TAGSΔΔTLR moiety from binding to the solid phase. This incubation of porcine sera in the presence of an excess of recombinant C-DIVA E2 molecules does not negatively influence the sensitivity of the assay but rather slightly increases the sensitivity (Fig. 3). This effect is unexpected and may be attributed to the capacity of recombinant C-DIVA E2 to absorb antibodies or other molecules interacting with E2 moieties in the proximity of TAVSPTTLR improving the chance of TAVSPTTLR specific antibodies to bind to the E2 on the solid phase. Taken together our data demonstrate that the new screening assay is at least comparable in sensitivity and specificity to the currently available commercial CSF assays. CDIVA combined with the new screening assay could be adopted as an alternative to the mandatory stamping out policy in Europe with its preventive killing of millions of pigs. Additionally the broad use of C-strain vaccines in Asia and South America could be replaced by vaccination with C-DIVA not only to avoid trade embargoes but also to eradicate the disease from endemic areas. Furthermore the novel assay principle described here may serve as a template for the generation of further marker vaccines for the control of other transboundary animal diseases. Acknowledgment We thank J. Kortekaas, W.L. Loeffen and S. Quak, (Virology Division, Central Veterinary Institute of Wageningen University Research Center, Lelystad, The Netherlands) for the access to their sera collections. Thanks also to Gerrit Korf and the Knapp team at the Free University Berlin for their support and for making available the facilities for the computational work. References Berzofsky, J.A., Schechter, A.N., 1981. The concepts of crossreactivity and specificity in immunology. Mol. Immunol. 18, 751. Brooks, B.R., Brooks III, C.L., Mackerell Jr., A.D., Nilsson, L., Petrella, R.J., Roux, B., Won, Y., Archontis, G., Bartels, C., Boresch, S., Caflisch, A., Caves, L., Cui, Q., Dinner, A.R., Feig, M., Fischer, S., Gao, J., Hodoscek, M., Im, W., Kuczera, K., Lazaridis, T., Ma, J., Ovchinnikov, V., Paci, E., Pastor, R.W., Post, C.B., Pu, J.Z., Schaefer, M., Tidor, B., Venable, R.M., Woodcock, H.L., Wu, X., Yang, W., York, D.M., Karplus, M., 2009. CHARMM: the biomolecular simulation program. J. Comput. Chem. 30, 1545.

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Please cite this article as: Bruderer, U., et al., Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating ..., J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.03.009

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

Serological differentiation between infection and vaccination depends on the detection of pathogen specific antibodies for an epitope that is modified...
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