aematopoietic depletion in vaccine-induced neonatal pancytopenia epends on both the titre and specificity of alloantibody and levels of HC I expression

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine’s production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells used in vaccine production, coupled with a powerful adjuvant, are responsible for the generation of pathogenic alloantibodies. ublis © 2015 The Authors. P . Introduction Bovine Neonatal Pancytopenia (BNP) is a disease of calves that merged in Europe from 2007 [1,2] and subsequently in New ealand [3]. It is characterised by profound depletion of periphral leukocytes and thrombocytes and bone marrow trilineage Abbreviations: BNP, Bovine neonatal pancytopenia; BVDV, Bovine Viral Diarhoea Virus; CDC assay, complement-dependent cytotoxicity assay; MDBK cells, adin-Darby Bovine Kidney cells; PRA, Panel Reactive Antibody. ∗ Corresponding author. Tel. : +44 0 131 651 9246; fax: +44 0 131 651 9105. E-mail address: charlotte.bell@roslin.ed.ac.uk (C.R. Bell). ttp://dx.doi. org/10.1016/j.vaccine. 2015.05. 069 264-410X/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article /). hed by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). hypoplasia, and is mediated by the ingestion of alloantibodies in colostrum [4–6]. BNP has been linked epidemiologically to vaccination of the dams of affected calves with a particular inactivated Bovine Viral Diarrhoea Virus (BVDV) vaccine (Pregsure BVD, Pfizer Animal Health) which incorporates a novel adjuvant formulation [7–9]. However the incidence of clinical BNP has been reported to be only 0.0016% per dose of vaccine sold [10]. This, together with variable alloantibody binding of individual sera to lymphocytes from different donor cows [11], suggests specificity for a highly polymorphic antigen. Alloantibodies in BNP-dams have been shown to recognise Major Histocompatibility Complex class I (MHC I) molecules [12–14], leading to the hypothesis that Pregsure contains bovine under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.