Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules
et al. (2014) Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal
Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules. PLoS ONE 9(10): e109239. doi:10.1371/journal.pone.0109239
Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules
Rahel Kasonta 0
Mark Holsteg 0
Karin Duchow 0
James W. Dekker 0
Klaus Cussler 0
Justin G. Bendall 0
Max Bastian 0
Aftab A. Ansari, Emory University School of Medicine, United States of America
0 1 Division of Veterinary Medicine, Paul-Ehrlich-Institut , Langen, Germany , 2 Fonterra Research and Development Centre , Palmerston North , New Zealand, 3 Landwirtschaftskammer Nordrhein-Westfalen, Referat 34 Tiergesundheit, Bonn , Germany
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. Crossreactive 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.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: The authors received no specific funding for this work. Funding for this work was provided by the Paul-Ehrlich-Institut and the Fonterra Research and
Development Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: JD and JB are employees of the FONTERRA Research and Development Center. The center belongs to FONTERRA ltd., the dairy company
that manufactures and produces colostrum powder for human consumption. All other authors have no competing interests. RK, KD, KC and MB are employed by
the independent governmental German Federal Institute for Vaccines and Biomedicines. This does not alter the authors adherence to PLOS ONE policies on
sharing data and materials.
During the past two decades, bovine colostrum has gained
increasing interest as a dietary supplement for human
consumption. Several studies have proposed beneficial effects for colostral,
antimicrobial antibodies , while other studies have postulated
that small molecules such as peptidic growth factors may have an
advantageous influence on gastrointestinal disorders .
Consequently, international food and pharmaceutical companies have
developed an array of different colostrum based products ranging
from sports food  to dietary supplements aiming to ameliorate
unspecified diarrhea in AIDS patients . Annual sales of dry
colostrum ingredients reached a volume of 2,600 tonnes with a
value of US$80 million in 2007 .
However, in 2006 a new hemorrhagic syndrome of bovine
neonates, Bovine Neonatal Pancytopenia (BNP) was first observed
in Central European countries. The syndrome affects newborn
calves and is characterized by a complete destruction of the red
bone marrow, pancytopenia, severe bleeding and high lethality.
The syndrome is triggered by ingestion of colostrum from dams
that have previously been vaccinated with PregSureBVD, a
strongly adjuvanted, inactivated vaccine against bovine viral
diarrhea virus (BVDV) [8,9]. Due to its particular composition
that combines substantial amounts of bioprocess impurities with a
very efficient adjuvant system, this vaccine induces high titres of
bovine MHC-I-specific alloantibodies . Transfer of
alloreactive, maternal antibodies via colostrum to a newborn calf
carrying the corresponding alloantigens leads to severe
pancytopenia . The pathoetiology of BNP is therefore similar to
human alloimmune diseases such as the Rhesus-Incompatibility
Syndrome or Neonatal Alloimmune Thrombocytopenia (NAIT),
except that such diseases involve transfer of alloimmune antibodies
to the fetus in utero rather than via breast milk to neonates.
Due to the compelling evidence for the causal role of PregSure
BVD vaccine in the induction of BNP, the European Medicines
Agency (EMA) suspended the marketing authorisation for
PregSure in 2010 . Shortly after the problem had been
identified in Europe, a number of BNP cases were observed in
New Zealand leading to a withdrawal of the vaccine from the New
Zealand market .
This animal disease, amongst calves, raises questions about the
safety of such colostral products for human consumption.
Therefore a joint study was initiated between the Fonterra
Research and Development Centre and the Paul-Ehrlich-Institute
(PEI) to clarify whether the New Zealand haemorrhagic cases
amongst calves were due to BNP and to assess whether the
colostrum of the respective dams contained antibodies capable of
cross-reacting with human cells.
Materials and Methods
1. Serum and dairy samples
Two sets of serum samples from 8 and 4
PregSureBVDvaccinated New Zealand BNP dams were obtained. During the
calving season 2011 these cows gave birth to calves, which
developed BNP within the first three weeks of life. Sera from
PregSureBVD vaccinated animals with no history of BNP in their
progeny (n = 79) and from non-vaccinated (n = 20) or alternatively
BVDV-vaccinated animals (n = 20) served as controls. In the herds
with BNP-affected calves, the vaccination programme consisted of
two doses given 46 weeks apart, followed by annual
administration of a booster dose. The animals sampled had received a
minimum of three vaccinations of PregSureBVD in the years
preceding the production of a BNP-affected calf. The majority of
cows was of Holstein-Frisian breed. In New Zealand some cows
were Holstein-Frisian Jersey crossbreeds. The collection of blood
samples from NZ dairy cows was being undertaken by
veterinarians as part of routine disease investigations. In addition, milk and
colostrum samples were obtained from 12 and six, respectively, of
the BNP dams. Individual colostrum samples investigated in this
study were obtained from one of the first four milkings after
parturition. Raw milk and colostrum samples from individual cows
were subjected to continuous-flow High Temperature Short Time
pasteurization by: using a peristaltic pump to pass small volumes
(600 ml in total) through a miniature plate heat exchanger (PHE),
fitted to a hot-water heating supply; then through a holding tube
with the peristaltic pump adjusted such that the flow rate provided
a residence time of 15 seconds; then past an electronic
temperature probe to ensure that the flow of hot water to the
initial PHE was adjusted such that the temperature of the liquid
within the far end of the holding tube was at least 72.6uC; and
finally through a second miniature PHE, fitted to a iced-water
cooling supply; and then discarding the first 250 mL portion that
emerged, prior to collecting each sample. Commercial lots of New
Zealand whole milk powder and two colostrum powders were also
obtained. For the production, milk and colostrum were
pasteurized and then spray dried to powder. The colostrum powder was
manufactured from the factory located in the region of New
Zealand, which had the greatest proportion of dairy herds using
PregSureBVD vaccination. The colostrum used to manufacture
colostrum powder was pooled from across all cows in a herd into
each farm vat, and then pooled from multiple herds farms vats. So
finished colostrum powder used in this study represents a
colostrum blend from several thousand cows. Although the first
eight milkings after parturition may consist of colostrum, for the
production of commercial colostrum powder only colostrum
collected from the first four milkings was used. One colostrum
powder batch was manufactured in New Zealand in 2011, prior to
the prohibition against PregSureBVD treatment, while the second
sample was manufactured in New Zealand in 2012 from colostrum
that had been sourced exclusively from non-PregSure-treated
cows. All samples were held at -70uC for long term storage. The
powders were weighed and 0.1 g each was dissolved in 1 ml
phosphate buffered saline (PBS, PEI), prepared fresh before each
analysis. Whole colostrum was centrifuged twice at 11,0006g
followed by 25,0006g to remove cell debris, and stored at 220uC.
For comparison, serum samples of six European BNP dams form
North-Rhine-Westphalia were included in the study. According to
the new German animal welfare legislation, the study was
announced to and approved by the competent authority (State
Office for Nature, Environment and Consumer Protection of
North-Rhine-Westfalia, LANUV Recklinghausen, Germany; ref.
2. Cell preparation and cell culture
Bovine leukocytes were prepared from whole blood of healthy
heifers by ammonium chloride lysis and Ficoll gradient
centrifugation as described elsewhere . Short-term T cell lines were
obtained from peripheral blood mononuclear cells (PBMCs) by
phytohaemagglutinin (PHA) stimulation as previously described
. The resulting polyclonal T cell lines are hereafter referred to
as lymphoblasts. Human lymphoblasts were prepared accordingly
from buffy coats of healthy blood donors. The buffy coats were
purchased from the German Red Cross Bloodbank, Frankfurt. All
donors have given informed consent to the medicinal, scientific or
pharmaceutical use of their blood or preparations thereof. Before
distribution the material is anonymized. To the customer no
information is disclosed about individual donors. This procedure
has been approved by the local ethics committee (Votum 329/10;
ethics committee; Goethe-University, Frankfurt).
The bovine kidney cell line used for the production of
PregSureBVD, hereafter referred to as BK cell line, was kindly
provided by Pfizer Animal Health . The cell line was tested to
be free of BVDV and was maintained according to manufacturers
instructions as previously described .
3. Flow cytometry
To examine samples for the presence of opsonising alloimmune
antibodies, flow cytometry analyses were carried out as previously
described . Briefly, up to 16105 BK cells or lymphoblasts were
re-suspended in PBS containing 0.5% fetal calf serum (FCS,
Gibco). Sera, colostrum or milk and also individually pasteurized
colostrum samples and reconstituted commercial milk and
colostrum powders were added to a final dilution of 1:5 (if not
stated otherwise) followed by one hour incubation at 4uC. Cells
were then washed twice with PBS containing 0.5% FCS and cell
surface bound bovine IgG detected using a FITC-conjugated
polyclonal sheep-anti-bovine IgG antibody (Invitrogen). Median
fluorescence intensity (MFI) of living cells as defined by
ForwardScatter (FSC)/Sideward-Scatter (SSC)-gating was determined for
each sample using a BD Lsr II Flow Cytometer (Figs 14) or a BD
Accuri C6 Flow Cytometer (Figs 56). The Accuri device uses a
different log scale to display fluorescence data, which results in a
baseline shift of two log scales between the two devices. To take
this into account, figures using Accuri data were given an adapted
scale (as indicated in the axis title, which now reads: MFI (6100)).
To isolate and identify BNP associated alloantigens on BK cells
or lymphoblasts immunoprecipitation (IP) was performed as
previously described . In summary, cells were surface labeled
with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) and
incubated with bovine serum samples. Cells were then solubilized
with Lysis Buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris
and 5 mM EDTA) in an ultrasonic bath. Solutions were clarified
by centrifugation (10,0006g, 10 min) and the immune complexes
precipitated with protein G-sepharose beads (GE Healthcare),
eluted with Loading Buffer containing 6 M urea, 62.5 mM Tris,
2% SDS, 10% glycerol and 0.025% bromophenol blue, and
subjected to SDS-PAGE under non-reducing conditions.
Proteins were blotted onto nitrocellulose membranes
(Whatman). Biotinylated cell membrane proteins were detected using a
Streptavidin-HRP conjugate (Dianova) and visualized using
enhanced chemoluminescence (ECL; GE Healthcare) reagents.
MHC-I molecules were visualized by developing the membranes
with an anti-bovine MHC I antibody (IL-A88; AbD Serotec) or an
anti-HLA I antibody (w6/32, kindly provided by Steffen Tenzer,
University of Mainz) followed by a secondary anti-murine-IgGHRP
5. Affinity purification of BNP alloantibodies
BNP alloreactive antibodies were affinity purified as previously
described . Sera of PregSureBVD vaccinated dams,
alternatively vaccinated animals, or non-immunized controls were diluted
1:10 in PBS and incubated with BK cells. The cells were
repeatedly washed with 0.9% sodium chloride. Cell surface-bound
antibodies were detached by treatment with ice-cold citrate buffer
(0.12 M sodium citrate, pH 2.5) for 15 minutes. After
centrifugation (10,0006g, 5 min) supernatants were harvested and
immediately treated with neutralization buffer (1 M Tris HCl, pH 9.0).
6. Flow cytometric detection of complement-activating antibodies
To assess the complement activating activity of opsonising
alloantibodies, we adopted a flow cytometry based,
complementdependent cytotoxicity assay . Briefly, 16105 human
lymphoblasts, re-suspended in PBS containing 0.5% FCS, were incubated
with heat inactivated (56uC, 30 minutes) bovine sera at a final
dilution of 1:5 and incubated for 45 minutes at 4uC. Complement
activation was assessed by adding active or heat-inactivated rabbit
complement, i.e. fresh rabbit serum. After 10 minutes at 37uC,
propidium iodide (PI, Sigma-Aldrich) was added, the samples were
Figure 3. PregSureBVD induced BNP alloantibodies cross-react with human lymphoblasts. (A) A panel of sixteen BNP dam sera was
tested by flow-cytometry for the presence of alloreactive antibodies that bind to the BK cell line. As controls sera from non-immunized, alternatively
vaccinated or PregSureBVD-immunized non-BNP dams were included. (B) The same serum panel was tested for cross-reactive binding to human
lymphoblasts. (C) From the entire serum panel BNP-associated alloantibodies were purified by affinity purification. The affinity purified alloantibodies
were again tested for cross-reactive binding to human lymphoblasts. Black bars indicate the MFI obtained with individual sera or the corresponding
placed on ice and immediately analysed by flow cytometry. The
cytometer settings were limited to run a fixed volume of 20 ml. For
each serum sample the number of living, PI-negative cells (N) after
incubation with active or heat-inactivated rabbit complement was
determined. The percentage of cytotoxicity was calculated
according to the following equation: specific Lysis = 100% x (1
Nactive complement/Ninactive complement).
7. Statistical analysis
Single-tailed, paired or unpaired, homoskedastic Students t test
was performed to test for significant differences between groups,
indicated by the p-value in the respective graphs (significance level
a = 0.05). Pearsons test was used to calculate correlation
1. New Zealand dams with BNP calves have high alloreactive antibody titres
During the 2011 calving season, several New Zealand farms
experienced haemorrhagic disorders in bovine neonates. To
determine whether BNP, the vaccine-induced alloimmune
syndrome that had previously been described only in Central Europe,
was the underlying cause, serum samples from affected dairy herds
and unaffected controls were collected and investigated for the
presence of alloreactive antibodies by flow cytometric assay.
Bovine lymphoblasts obtained from an adult Holstein-Frisian cow
were incubated with respective sera and cell surface-bound
alloantibodies quantified by flow cytometry. Living cells were
identified according to their FSC/SSC characteristics and median
fluorescent intensity (MFI) was determined for the living cell
population (Fig S1). The MFI served as a correlate for the amount
of alloantibodies present in each serum sample. MFI obtained
from New Zealand BNP dam sera was similar to the confirmed
BNP cases from German dams (Fig 1A). In accordance with
previous observations made in Central Europe , we found that
BNP dams had significantly higher levels of alloantibodies than
PregSureBVD vaccinated animals giving birth to calves that did
not develop BNP, or animals that received an alternative BVDV
vaccine, or non-vaccinated controls (Fig 1A).
To confirm that bovine MHC-I molecules were targeted by the
vaccine induced alloantibodies, as previously described [11,12,16],
BNP dam sera were tested by immune precipitation. BK cells (i.e.
the cell line used in the production of PregSureBVD) were
biotinylated and incubated with each test sera. Cell surface
molecules complexed to the alloantibodies were precipitated,
separated by SDS-PAGE, blotted and visualized by
peroxidaseconjugated streptavidin (data not shown) or MHC-I-specific
staining. In parallel, the same samples were tested against BK
cells by flow cytometry. The level of alloantibody binding as
measured by flow cytometry correlates significantly with the
amount of bovine MHC-I detected, as revealed by the presence of
a Western blot band of approximately 43 kDa (Fig 1B and Fig
S2A and S3). However, in two out of the 12 BNP dam sera, no
alloantibody binding was detectable. Whether this is due to serum
degradation, or whether these calves suffered from BNP-like
symptoms not related to PregSureBVD induced MHC-I
alloantibodies, is not clear.
2. BNP associated alloantibodies are concentrated in colostrum
Bovine colostrum represents an antibody concentrate that
supplies the neonate with maternal antibodies, and it has been
shown that BNP-associated alloantibodies are present in the
colostrum of BNP dams . To assess the amount of
BNPassociated alloantibodies in colostrum and milk, matched samples
of: colostrum, serum and milk from six individual New Zealand
BNP dams were tested. We found that the reactivity to BK cell
surface molecules was highest in colostrum, followed by serum,
while only low to negligible amounts of BNP-associated
alloantibodies were present in milk. Presumably, due to one outlier the
difference between colostrum and serum is not significant (Fig 2).
To test whether pasteurization potentially reduces the activity of
colostrum contained alloantibodies, individually pasteurized
colostrum samples were compared to unpasteurized colostrum
samples. However, no significant change in binding capacity of the
alloantibodies could be detected (data not shown). Colostrum is
only produced during the first days after birth, whereas the first
clinical signs of BNP occur ten days after birth making it
challenging to obtain colostrum from BNP dams. Since the
alloreactivity was sufficiently detectable in both colostrum and
serum, we performed the majority of the subsequent experiments
on serum samples.
3. BNP-associated alloantibodies cross-react with human lymphoblasts
To investigate whether BNP-associated alloantibodies
crossreact with human cells, we compared the surface-binding
reactivity of our serum panel to the BK cell line with human
lymphoblasts. There was a significant correlation (R = 0.71) of the
reactivity to BK cells and to human lymphoblasts (Fig 3A and B).
To confirm that this reactivity was due to PregSureBVD
vaccination, the experiment was repeated using affinity-purified,
vaccine-induced alloantibodies. While affinity-purified
alloantibodies from PregSureBVD-vaccinated animals, i.e. three out of
eight PregSureBVD immunized non-BNP and thirteen out of
sixteen BNP dams, exhibited the same cross-reactivity pattern to
human lymphoblasts as the sera, the minor reactivity seen with
sera from animals not vaccinated with PregSureBVD disappeared
indicating that those signals were non-specific. To identify the
antigen BNP-associated alloantibodies recognized on human
lymphoblasts, we performed immunoprecipitation experiments
using human lymphoblasts. Again, for BNP dam sera the flow
cytometric reactivity correlated significantly with the precipitation
of human MHC I (Fig 4 and Fig S2B), while sera from
nonimmunized or alternatively vaccinated animals displayed no MHC
I-specific reactivity (data not shown).
It has been speculated that complement-mediated lysis of
alloantibody-opsonised cells contributes to the pathogenesis of
BNP [10,18]. To assess whether BNP-associated alloantibodies
could potentially destroy human lymphocytes through such a
mechanism, we adapted a flow-cytometric approach to measure
complement activity in vitro . All ten BNP sera that contained
alloreactive antibodies induced a specific complement-lysis, while
the corresponding control sera showed no effect (Fig 5). This
indicates that BNP-associated alloantibodies not only recognize
human MHC-I molecules, but also have the capacity to exert a
cytotoxic effect on human lymphoblasts. The same observation was
made on BK cells with PregSureBVD induced, MHC-I reactive
alloantibodies from non-BNP and BNP dams (Fig S3 and S4).
4. BNP Alloantibodies are present in commercially available dairy products
To test whether BNP-associated alloantibodies are present
in commercial lots of colostrum powder, the material used by
down-stream industries to produce colostrum based dietary
supplements for human consumption, we investigated milk and
colostrum powders that had been produced during and after the
2011 calving season. Flow cytometry showed significant reactivity
against BK cells and human lymphoblasts in the 2011 colostrum
powder lot (Fig 6), although due to a production-inherent dilution
effect the binding is much lower compared to the reactivity of
individual BNP dam colostra (see Fig 2 for comparison). In view of
this observation, Fonterra withheld all 2011 colostrum from sale,
and in 2012 changed the colostrum harvesting policy to exclude all
animals that had been vaccinated with PregSureBVD. Colostrum
powder manufactured during the 2012 calving season displayed no
cross-reactivity to BK cells or human lymphoblasts.
The aim of the current study was to clarify whether cases of
bovine neonate haemorrhages in New Zealand were caused by the
vaccine induced feto-maternal incompatibility syndrome, BNP.
Furthermore, the question was addressed whether the
haemorrhage causing alloantibodies in the colostrum of PregSureBVD
immunized cows could cross-react with human cells and might
therefore pose a theoretical risk to human consumers of products
manufactured from such colostrum.
The data presented in the current study are consistent with the
contention that the cases of bovine neonate haemorrhage observed
in NZ were caused by BNP. The alloreactivity in sera of NZ dairy
cows showed the same reactivity pattern as that previously
observed in Europe [10,13]. Only sera from PregSureBVD
immunized animals showed alloreactive binding to bovine
lymphocytes, and this alloreactivity was directed against MHC-I
molecules of BK cells. Although this alloreactivity was present in
the serum, it was clearly higher in the colostrum of BNP dams.
This is in concordance with observations in Europe and was
expected because, in ruminants, colostrum is the only source of
maternal antibodies and contains high amounts of
immunoglobulins . Compared to serum or colostrum, the level of
alloantibodies in milk was negligible. From this observation there
is no evidence that the consumption of milk from BNP dams is
hazardous for human consumers.
By contrast, for colostrum or serum our data clearly prove that
BNP-associated alloantibodies cross-react with human MHC-I
molecules. Whether this could potentially be hazardous for human
consumers, cannot be conclusively answered in this study: In
humans, alloimmune syndromes with severe clinical signs are
generally attributed to specific alloantigens that are expressed on
particular target cell types only. For example, NAIT is induced in
most cases by alloantibodies targeting a dimorphic epitope in the
beta chain of Human Platelet Antigen 1 (HPA-1a), which is only
present on megakaryocytes and thrombocytes . The particular
expression pattern of the alloantigen explains the specific insult of
NAIT antibodies on the haemostatic system . Although
MHCI specific antibodies have originally been implicated in the
induction of NAIT, it was later shown that they rather play an
inferior role . In the case of BNP no specific target antigen
other than MHC-I has yet been identified. Instead, there is
evidence for a causal role of MHC-I specific alloantibodies.
Several studies have identified MHC-I specificity as the only
common denominator in the serum of BNP dams [11,12,16,21].
Furthermore, Bridger et al. demonstrated in an elegant study that
the surface opsonising activity of orally administered BNP dam
antibodies which has been shown to correlate with MHC-I
reactivity is directly associated with the severity of clinical
symptoms in challenged calves . Finally, we observed in two
cases that fraternal twins of BNP-affected calves, expressing
MHCI molecules that were not recognized by the alloantibodies of the
dam, survived the ingestion of toxic colostrum and exhibited a
completely normal appearance of their red bone marrow . So,
one important current hypothesis is that MHC-I specific
alloantibodies cause BNP in bovines.
However, despite the complement activating effect on human
lymphoblasts observed in vitro, it is questionable whether the
bovine alloantibodies could cause any effect in humans. The main
difficulty with a pathoetiological role of MHC-I-specific antibodies
is that MHC-I proteins are ubiquitously expressed on the vast
majority of cells. Even though the actual pathomechanism leading
to cell destruction after alloantibody intake is not elucidated, it can
be assumed that large amounts of MHC-I alloantibodies would be
required to saturate peripheral MHC-I before such antibodies
could reach a tissue and induce a specific insult. It is conceivable
that with the sudden uptake of several grams of maternal
antibodies in calves, such levels of alloantibodies are reached.
However, it is improbable for such a massive influx to occur in
humans. Firstly, in contrast to bovine neonates, bovine dietary
antibodies are not effectively transported across the human gut
mucosa. The neonatal uptake of maternal antibodies via the gut
mucosa is mediated by the neonatal Fc-receptor, FcRn, a
dedicated transport system that shuttles maternal antibodies across
the gut-blood barrier. It is known that the mucosal barrier closes
2448 hrs after birth and at least in rodents it has been shown that
FcRn is downregulated directly after birth . In humans, the
fetus is provided with maternal antibodies via the placenta during
gestation [24,25]. While the neonatal Fc-receptor is expressed
mainly in the fetal placenta, it is also found in the gut mucosa 
where it is expressed throughout life [27,28]. In vitro studies have
shown that whilst the expressed receptor is functional and
transports antibodies across polarized cell layers , the main
function of human FcRn-expression in the gut is to shuttle
antibodies from the submucosal side into the gut lumen . The
direction of the net transport is determined by the IgG
concentration gradient . Since the affinity of the human FcRn
for human IgG is four-fold higher than that of bovine IgG ,
and since the concentration of cross-reactive alloantibodies in
commercial colostrum products is relatively low, the uptake of
cross-reactive alloantibodies is inefficient, such that only
insignificant amounts of MHC-I-specific BNP antibodies would reach the
Secondly, there are cases where human breast milk can contain
alloreactive antibodies. For instance, for NAIT, platelet-specific
antibodies have been identified in the breast milk of affected
mothers, but this breast milk is still considered safe to consume by
the infant . Similarly for other maternal autoimmune diseases,
such as lupus or Hashimotos thyroiditis, disease manifestation in
the infant is due to maternal alloantibodies transferred in utero
rather than via lactation, such that disease exacerbation is not
associated with breast feeding [33,34]. This suggests that
alloantibodies present in the infant diet are unlikely to result in
detrimental health effects.
Taking all these observations and considerations together, we
conclude that the risk of colostrum-containing dietary
supplements, from cows treated with the vaccine associated with BNP, is
low for human consumers. However, since the reactivity of BNP
alloantibodies to human lymphoblasts is principally
indistinguishable from the reactivity to bovine cells, a residual risk cannot be
absolutely excluded. In what may have been an overly cautious
approach, Fonterra decided to withhold the entire colostrum
production of 2011, and from 2012 onwards excluded colostrum
collection from PregSure-treated cows. This approach was
effective, as no alloreactivity to bovine or human cells was
detectable in colostrum powder manufactured according to the
new supply policy.
Figure S1 The gating strategy of the flow-cytometric
analysis. BK cells (A) and bovine (B) or human lymphoblasts (C)
were incubated with serum from a non.-immunized cow
(punctuate line) or a BNP dam (bold line). Cells were washed
twice and incubated with a FITC-conjugated secondary antibody.
Subsequently, cells were analyzed by flow-cytometry. Live cells
were identified and gated according to their FSC/SSC
characteristics (black lined area in the left panels). The median fluorescence
intensity was analyzed for all events within the live cell gate (right
Figure S2 Alloantibody reactivity measured by FACS
correlates with MHC-I immunoprecipitation. The
intensity of immunoprecipitated MHC-I bands shown in Fig 1B and
Fig 4 was analyzed by densitometry. The arbitrary grey value of
the densitometry was plotted against the corresponding
alloantibody binding as determined by flow-cytometry (MFI). The
correlation is shown for BK cells (A) and for human lymphoblasts
(B). Pearson correlation coefficients and p-values are indicated.
Figure S3 Alloantibody reactivity measured by FACS
correlates with MHC-I immunoprecipitation. (A) BK cells
were incubated with eight sera of non-immunized or alternatively
BVD-vaccinated cows or of PregSureBVD-vaccinated non-BNP
dams and PregSureBVD vaccinated BNP dams. Surface molecules
were immunoprecipitated as described. The precipitates were
analyzed by SDS-PAGE followed by anti BoLA-I westernblot
using monoclonal antibody IL A88. Specific MHC-I bands at
about 40 kDa (lined area) were analyzed by densitometry. (B) The
same panel of sera was tested in parallel by flow-cytometry for
alloantibody binding. MFI values (black bars) are plotted side by
side with the corresponding grey values as determined by
densitometry (grey bars). The correlation was calculated, the
respective Pearson coefficient and the p-value are indicated.
Figure S4 Alloantibody mediated complement lysis
correlates with FACS reactivity. (A) BK cells were incubated
with serum from a non-immunized cow (left panel) or a BNP dam
(right panel). Heat-inactivated (dotted line) or active rabbit
complement (bold line) was added and samples were incubated
at 37uC. To identify dead cells red fluorescent propidium iodide
was added and samples were analyzed by FACS. The absolute
number of living, propidium iodide negative cells in a defined
sample volume of 20 ml was determined. Numerical figures in the
graphs represent the respective number of living cells after adding
active complement. (B) Specific cell lysis was determined for the
same serum panel as in Fig S3 and tested in parallel by
flowcytometry for alloantibody binding. MFI values (black bars) are
plotted side by side with the corresponding %-specific cell lysis
(open bars). The correlation was calculated, the respective Pearson
coefficient and the p-value are indicated.
We are grateful for the technical support of H. Hanke-Robinson and we
appreciate the contribution of E. Schwedinger and the staff from the PEI
Conceived and designed the experiments: RK MH JD JB MB. Performed
the experiments: RK MB. Analyzed the data: RK MH JD JB MB.
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