Karin Duchow

Bovine Neonatal Pancytopenia: Is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies?

Abstract Bovine Neonatal Pancytopenia (BNP) is a new emerging disease observed since 2007 in Germany and neighbouring countries. The syndrome affects newborn calves and is characterized by pancytopenia, severe bleeding and high lethality. So far, a causative role of infectious or toxic agents has been ruled out. Instead, the syndrome is induced after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In analogy to similar diseases in humans it has therefore been postulated that BNP is caused by alloreactive, maternal antibodies. There is a striking association between BNP and a previous vaccination of the respective dams with a particular vaccine against Bovine Virus Diarrhoea (BVD). This association has led to a suspension of the marketing authorisation for the vaccine, by the European Commission. The current study investigates the role of this vaccine in the pathogenesis of BNP. By flow cytometry we were able to demonstrate that sera of BNP dams (dams that gave birth to a BNP calf) harbour alloreactive antibodies binding to surface antigens on bovine leukocytes. A significantly weaker alloreactivity was observed with sera of non-BNP dams that have been vaccinated with the same vaccine but delivered healthy calves. No binding was seen with non-BVD-vaccinated control cows and animals that were vaccinated with other inactivated BVD vaccines so far not associated with BNP. The binding is functionally relevant, because opsonization of bovine leukocytes with alloantibodies led to an elevated cytophagocytosis by bovine macrophages. To test whether the vaccine induces alloreactive antibodies two strategies were employed: Guinea pigs were vaccinated with a panel of commercially available BVD-vaccines. Only the incriminated vaccine induced antibodies binding surface antigens on bovine leukocytes. Additionally, two calves were repeatedly vaccinated with the suspected vaccine and the development of alloreactivity was monitored. In dependence of the number of booster immunizations the induction of alloreactive antibodies could be observed. Finally, by affinity purification we were able to directly demonstrate that BNP associated alloantibodies cross react with the bovine kidney cell line used for vaccine production. Together this provides strong evidence that this particular BVD vaccine has the potential to induce BNP associated alloantibodies.

The rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines.

The European Pharmacopoeia proposes two methods for potency determination of inactivated rabies vaccines for veterinary use: The first one is a classical mouse challenge test, which is imprecise, time-consuming, and causes severe distress to the test animals. Alternatively, the potency may be determined serologically by measuring the neutralizing antibody titers induced after vaccination of mice by using a rapid fluorescent focus inhibition test (RFFIT). Although this method is faster and less painful for the animals, it is not widely used yet, and only little data exist concerning the comparability of both methods. We have therefore performed a comparative study, in which we demonstrated a good correlation between the challenge test results and the mean titers determined by RFFIT. Furthermore, all vaccine batches failing the challenge test were also recognized as insufficient in the serological assay. This publication further describes the influence of different vaccine administration routes on the resulting antibody titers, and it proposes various modifications to the serological assay protocol which could improve its overall practicability. Finally, we recommend that the serological assay be used for the potency testing of inactivated rabies vaccines.

Vaccination against Feline Panleukopenia: implications from a field study in kittens

BackgroundFeline Panleukopenia (FPL) is a serious disease of cats that can be prevented by vaccination. Kittens are routinely vaccinated repeatedly during their first months of life. By this time maternally derived antibodies (MDA) can interfere with vaccination and inhibit the development of active immunity. The efficacy of primary vaccination under field conditions was questioned by frequent reports to the Paul-Ehrlich-Institut on outbreaks of FPL in vaccinated breeding catteries. We therefore initiated a field study to investigate the development of immunity in kittens during primary vaccination against FPL.64 kittens from 16 litters were vaccinated against FPL at the age of 8, 12 and 16 weeks using three commercial polyvalent vaccines. Blood samples were taken before each vaccination and at the age of 20 weeks. Sera were tested for antibodies against Feline Panleukopenia Virus (FPV) by hemagglutination inhibition test and serum neutralisation assay in two independent diagnostic laboratories.ResultsThere was a good correlation between the results obtained in different laboratories and with different methods. Despite triple vaccination 36.7% of the kittens did not seroconvert. Even very low titres of MDA apparently inhibited the development of active immunity. The majority of kittens displayed significant titres of MDA at 8 and 12 weeks of age; in some animals MDA were still detected at 20 weeks of age. Interestingly, the vaccines tested differed significantly in their ability to overcome low levels of maternal immunity.ConclusionsIn the given situation it is recommended to quantify antibodies against FPV in the serum of the queen or kittens before primary vaccination of kittens. The beginning of primary vaccination should be delayed until MDA titres have declined. Unprotected kittens that have been identified serologically should be revaccinated.

Effect of the vaccination scheme on PregSure® BVD induced alloreactivity and the incidence of Bovine Neonatal Pancytopenia.

Bovine Neonatal Pancytopenia (BNP) is a new neonate-maternal incompatibility phenomenon caused by vaccine-induced, maternal alloantibodies. The syndrome affects newborn calves at the approximate age of ten days and is characterized by spontaneous bleeding, severe anemia with an almost complete destruction of the red bone marrow. During the past two years the causal role of bioprocess impurities in PregSure(®)BVD, a strongly adjuvanted, inactivated vaccine against Bovine Virus Diarrhoea (BVD), in the induction of BNP causing alloantibodies has clearly been established. Despite intensive research efforts that have elucidated the basic principles of the BNP immunopathology still a number of questions remain open. In the current manuscript we address the puzzling observation that BNP incidences vary widely between different regions: as an example we compare the BNP incidences in the German Federal States of Bavaria and Lower Saxony. In Bavaria the BNP-incidence reaches 100 cases per 100,000 doses PregSure(®)BVD, while in Lower Saxony the incidence is as low as 6 cases per 100,000 doses. In Bavaria the vaccine has always been used according to the instructions for use. By contrast, in Lower Saxony BVD-immunization was performed according to a two-step vaccination protocol including a first immunization with an inactivated BVD-vaccine followed by booster immunizations with a live-attenuated BVD-vaccine. As a consequence, those cattle that received PregSure(®)BVD received in general more than two doses in Bavaria, while in Lower Saxony cows received at maximum one dose. By experimental immunization we can show that the two-step regimen including PregSure(®)BVD as a priming vaccine results in significantly lower alloantibody titers as compared to repetitive immunizations with the inactivated vaccine. The lower alloantibody titer after two-step vaccination explains the lower BNP-incidence in Lower Saxony and - generally speaking - indicates that variations in the vaccination regimen have a great influence on the induction of adverse reactions through bioprocess impurities.

High definition viral vaccine strain identity and stability testing using full-genome population data--The next generation of vaccine quality control.

BACKGROUND Vaccines are the most effective prophylactic public health tools. With the help of vaccines, prevention of infectious disease spread and, in concert with other measures, even eradication has become possible. Until now, licensing and quality control require the determination of consensus genome sequences of replication competent infectious agents contained in vaccines. Recent improvements in sequencing technologies now enable the sequencing of complete genomes and the genetic analysis of populations with high reliability and resolution. The latter is particularly important for RNA viruses, which consist of fluctuating heterogeneous populations rather than genetically stable entities. This information now has to be integrated into the existing regulatory framework, challenging both licensing authorities and vaccine producers to develop new quality control criteria. METHODS Commercially available modified-live oral rabies vaccines and their precursor strains were deep-sequenced to assess strain identity and relations between strains based on population diversity. Strain relations were inferred based on the Manhattan distances calculated between the compositions of the viral populations of the strains. RESULTS We provide a novel approach to assess viral strain relations with high resolution and reliability by deep sequencing with subsequent analysis of the overall genetic diversity within the viral populations. A comparison of our novel approach of inferring strain relations based on population data with consensus sequence analysis clearly shows that consensus sequence analysis of diverse viral populations can be misleading. Therefore, for quality control of viral vaccines deep sequencing analysis is to be preferred over consensus sequence analysis. CONCLUSIONS The presented methodology allows for routine integration of deep sequencing data in vaccine quality control and licensing for highly reliable assessment of strain identity and stability.

A multi-dose serological assay suitable to quantify the potency of inactivated rabies vaccines for veterinary use

The mouse vaccination-challenge test, which is the most widely used method for determining the potency of inactivated rabies vaccines, is imprecise, time-consuming, and causes severe distress to the test animals. An alternative single-dose serological method has been implemented in the European Pharmacopoeia Monograph 0451 to replace the mouse challenge test for batch release. This single-dose limit method provides semi-quantitative results, but is not suitable for quantifying potency. We have now extended this serological method to a multi-dose format which allows a quantification of vaccine potency. In studies including all rabies vaccine strains relevant for Europe, we found dose-dependency for all vaccines and standard preparations. We have demonstrated that the multi-dose serological approach provides reliable quantitative potency results and is more precise than the mouse vaccination-challenge test. We have shown that adjuvanted vaccines can be calibrated against non-adjuvanted material, and that reference material can be calibrated against the International Standard. The method is therefore capable of assigning potency with the additional advantage of requiring fewer animals and reducing distress. Once the applicability of the method has been further verified in a collaborative study, it can complement the single-dose assay and eventually eliminate the need for the mouse challenge test.

A new lymphocyte proliferation assay for potency determination of bovine tuberculin PPDs.

The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. Aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future. To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.

Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules

In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP), was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf) cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.

Bovine Neonatal Pancytopenia-Associated Alloantibodies Recognize Individual Bovine Leukocyte Antigen 1 Alleles

Bovine neonatal pancytopenia (BNP) was a vaccine-induced alloimmune disease observed in young calves and characterized by hemorrhages, pancytopenia, and severe destruction of the hematopoietic tissues. BNP was induced by alloreactive maternal antibodies present in the colostrum of certain cows vaccinated with a highly adjuvanted vaccine against bovine viral diarrhea. Bioprocess impurities, originating from the production cell line of the vaccine, are likely to have induced these alloreactive antibodies. One prominent alloantigen recognized by vaccine-induced alloantibodies is highly polymorphic bovine major histocompatibility complex class I antigen (bovine leukocyte antigen 1—BoLA I). Aim of this study was to define the fine specificity of BNP-associated anti-BoLA I alloantibodies. In total, eight different BoLA I alleles from the production cell line were identified. All genes were cloned and recombinantly expressed in murine cell lines. Using these cells in a flow cytometric assay, the presence of BoLA I specific alloantibodies in BNP dam sera was proven. Three BoLA I variants were identified that accounted for the majority of vaccine-induced BoLA I reactivity. By comparing the sequence of immunogenic to non-immunogenic BoLA I variants probable minimal epitopes on BoLA I were identified. In general, dams of BNP calves displayed high levels of BoLA I reactive alloantibodies, while vaccinated cows delivering healthy calves had significantly lower alloantibody titers. We identified a subgroup of vaccinated cows with healthy calves displaying very high alloantibody titers. Between these cows and BNP dams no principle difference in the BoLA I reactivity pattern was observed. However, with a limited set of dam-calf pairs it could be demonstrated that serum from these cows did not bind to BoLA I expressing leukocytes of their offspring. By contrast, when testing cells from surviving BNP calves with the corresponding dam’s serum there was significant binding. We therefore conclude that predominantly highly alloreactive cows are at risk to induce BNP and it depends on the paternally inherited BoLA I whether or not the calf develops BNP.

Regulatorische Anforderungen an Zelltherapeutika in der Human- und in der Veterinärmedizin – ein Vergleich@@@Regulatory requirements regarding cell-based medicinal products for human and veterinary use - a comparison

At present, there is no separate regulatory framework for cell-based medicinal products (CBMP) for veterinary use at the European or German level. Current European and national regulations exclusively apply to the corresponding medicinal products for human use. An increasing number of requests for the regulatory classification of CBMP for veterinary use, such as allogeneic stem cell preparations and dendritic cell-based autologous tumour vaccines, and a rise in scientific advice for companies developing these products, illustrate the need for adequate legislation. Currently, advice is given and decisions are made on a case-by-case basis regarding the regulatory classification and authorisation requirements.Since some of the CBMP - in particular in the area of stem-cell products - are developed in parallel for human and veterinary use, there is an urgent need to create specific legal definitions, regulations, and guidelines for these complex innovative products in the veterinary sector as well. Otherwise, there is a risk that that the current legal grey area regarding veterinary medicinal products will impede therapeutic innovations in the long run. A harmonised EU-wide approach is desirable. Currently the European legislation on veterinary medicinal products is under revision. In this context, veterinary therapeutics based on allogeneic cells and tissues will be defined and regulated. Certainly, the legal framework does not have to be as comprehensive as for human CBMP; a leaner solution is conceivable, similar to the special provisions for advanced-therapy medicinal products laid down in the German Medicines Act.