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

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

BACKGROUND: The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. METHODS: We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. RESULTS: No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. CONCLUSIONS: In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.)

On the aetiology of bovine neonatal pancytopenia (BNP)

Bovine Neonatal Pancytopenia (BNP) is a novel haemorrhagic disease in sucking calves, characterised by bleeding, haematological changes and high mortality. Dams that gave birth to BNP affected calves were immunized with PregSure® BVD, a highly adjuvanted vaccine against Bovine Viral Diarrhoea (BVD). We can show that bioprocess impurities in the vaccine, originating from the cell line used for vaccine production induces alloantibodies in vaccinated cattle. Via flow cytometry and immunoprecipitation we can demonstrate that PregSure® BVD immunization leads to BNP alloantibody production. BNP alloantibodies target highly polymorphic bovine MHC-I molecules (BoLA I). We sequenced eight BoLA I variants expressed by the production cell line and identified three alleles which are responsible for the majority of PregSure® BVD induced BoLA I reactivity. The BoLA I alleles of BNP unaffected calves are not recognized by the BNP associated alloantibodies of their respective dams. We also examined whether BNP alloantibodies cross-react with human cells, thus being a potential hazard for human colostrum consumers and could show that BNP alloantibodies are cross-reactive to human MHC-I and can even be found in commercial colostrum powder manufactured from cows immunized with PregSure® BVD. Overall we can demonstrate that BNP is a vaccine induced alloimmune disease.Die Bovine Neonatale Panzytopenie (BNP) ist ein neuartiges hämorrhagisches Krankheitsbild bei Saugkälbern, das mit Blutungsneigung, hämatologischen Veränderungen und einer hohen Letalität einhergeht. Mutterkühen erkrankter Kälber wurden mit PregSure® BVD, ein stark adjuvantierter Impfstoff gegen Bovine Virusdiarrhoe (BVD) immunisiert. Der Impfstoff enthält Zellbestandteile von der Zellinie die zur Virusproduktion eingesetzt wird. Diese Zellbestandteile, führen bei geimpften Kühen zu der Bildung alloreaktiver BNP assoziierte Antikörper. Mittels Durchflusszytometrie und Immunpräzipitation konnten wir zeigen, dass PregSure® BVD Immunisierung zu einer BNP Alloantikörper Produktion führt. BNP Alloantikörper sind gegen hoch polymorphe Rinder MHC-I-Moleküle gerichtet (BoLA I). Acht BoLA I-Varianten aus der Produktionszelllinie wurden isoliert und davon wurden drei Allele identifiziert, die für die Mehrheit der PregSure® BVD induzierte BoLA I Reaktivität verantwortlich sind. Die BoLA I-Varianten von gesunden Kälbern werden nicht von den BNP assoziierten Alloantikörpern ihrer jeweiligen Muttertiere erkannt. Weiterhin haben wir untersucht ob BNP Alloantikörper mit menschlichen Zellen kreuz reagieren, um eine potenzielle Gefahr für Verbraucher von Rinderkolostrum auszuschließen. Wir konnten nachweisen, dass BNP Alloantikörper auch menschliche MHC-I Moleküle binden. BNP assoziierte Alloantikörper befinden sich auch in kommerziell hergestelltem Kolostrum Pulver, produziert aus Kolostrum von PregSure® BVD immunisierten Kühen. Zusammenfassend können wir zeigen, dass BNP ein Impfstoff induziert alloimmune Krankheit ist

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

Sera of NZ-BNP dams show identical alloreactivity compared to European BNP dams.

<p>(A) Sera were obtained from NZ dairy cows that had either not been immunized against BVDV (non-BVD-imm.), vaccinated with an alternative BVD vaccine (BVD-vaccine), vaccinated with PregSureBVD and birthed healthy calves (PregSure) or cows that had been vaccinated with PregSureBVD and gave birth to BNP calves (BNP dams (NZ)). By flow cytometry a first set of eight sera per group was tested for alloreactive binding to bovine lymphoblasts. As a control we included six serum samples of European BNP dams (BNP dam (EUR)). Symbols represent the median fluorescence intensity (MFI) for individual serum samples, black bars indicate the median value for each group. (B) Using BK cells, <i>i.e</i>. the cell line used for the production of PregSureBVD, the full panel of serum samples from twelve NZ BNP dams were tested in parallel by flow cytometry and immunoprecipitation for the presence of BNP associated alloantibodies. The upper panel shows the MFI for the individual serum samples, the lower panel shows the corresponding immunoprecipitate as revealed by a monoclonal antibody specific for bovine MHC-I molecules. Serum from a European BNP dam served as a positive control, fetal calf serum was used as a negative control.</p

BNP-associated alloantibodies bind human MHC-I molecules.

<p>The panel of BNP dam sera was tested in parallel by flow-cytometry and by immunoprecipitation using human lymphoblasts. The black bars in the upper panel show the MFI as determined by flow cytometric analysis. The lower panel shows the corresponding immunoprecipitates as revealed by a monoclonal antibody specific for human MHC-I molecules.</p

BNP-associated alloantibodies opsonise and sensitize human lymphoblasts for complement-mediated cell lysis.

<p>Human lymphoblasts were coincubated with heat-inactivated bovine serum and rabbit complement was added. Cell viability was measured by flow cytometry counting live, propidium-iodide negative cells. (A) The histograms show human lymphoblasts incubated with serum from a non-PregSureBVD immunized control dam (left) or BNP dam serum (right) after adding active complement (bold line). As a control, heat inactivated rabbit complement was added (dotted line). Numerical figures indicate the absolute number of living, propidium-iodide negative cells. (B) The serum panel was tested for its complement sensitizing activity on human lymphoblasts. Sera from four non-immunized and four alternatively vaccinated animals served as a control. One representative of three experiments is shown. Black bars represent the specific lysis for the individual sera.</p

Exclusion of PregSureBVD immunized animals reduces the amount of cross-reactive antibodies in commercial colostrum powder.

<p>Two commercial lots of colostrum powder were compared by flow cytometry to whole milk powder for the presence of BNP-associated alloantibodies, one lot was produced during the calving season of 2011, the other was produced in 2012 according to a new harvesting policy excluding herds that had been vaccinated with PregSurerBVD. Filled circles represent the MFI for colostrum powder produced in 2011, open circles for 2012 colostrum, open quadrates for whole milk powder. Panel (A) depicts the reactivity for BK cells, panel (B) the reactivity to human lymphoblasts. Symbols represent the median over three independent flow cytometric analyses, error bars the corresponding standard deviation. Asterisks indicate a significant difference between the two colostrum batches at the indicated dilution. For the analyses an Accuri C6 flow cytometer was used.</p

Systems Vaccinology Identifies an Early Innate Immune Signature as a Correlate of Antibody Responses to the Ebola Vaccine rVSV-ZEBOV

International audiencePredicting vaccine efficacy remains a challenge. We used a systems vaccinology approach to identify early innate immune correlates of antibody induction in humans receiving the Ebola vaccine rVSV-ZEBOV. Blood samples from days 0, 1, 3, 7, and 14 were analyzed for changes in cytokine levels, innate immune cell subsets, and gene expression. Integrative statistical analyses with cross-validation identified a signature of 5 early innate markers correlating with antibody titers on day 28 and beyond. Among those, IP-10 on day 3 and MFI of CXCR6 on NK cells on day 1 were independent correlates. Consistently, we found an early gene expression signature linked to IP-10. This comprehensive characterization of early innate immune responses to the rVSV-ZEBOV vaccine in humans revealed immune signatures linked to IP-10. These results suggest correlates of vaccine-induced antibody induction and provide a rationale to explore strategies for augmenting the effectiveness of vaccines through manipulation of IP-10