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# Development of a bovine/ovine cytokine 15-plex assay for immunoprofiling of the cellular response in ruminants

Jérémy Lesueur, Sarah Barbey, Nathan Cebron, Sarah Walachowski, Rachel

Lefebvre, Pierre Germon, Didier Boichard, Fabien Corbière, Gilles Foucras

► To cite this version:

Jérémy Lesueur, Sarah Barbey, Nathan Cebron, Sarah Walachowski, Rachel Lefebvre, et al. Development of a bovine/ovine cytokine 15-plex assay for immunoprofiling of the cellular response in ruminants. 12. International Veterinary Immunology Symposium, Aug 2019, Seattle, United States. 176 p. hal-02738448

HAL Id: hal-02738448

https://hal.inrae.fr/hal-02738448

Submitted on 2 Jun 2020

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ABSTRACT BOOK

International Veterinary
Immunology Symposium

AUG 13 – 16, 2019 | RENAISSANCE SEATTLE, SEATTLE, USA
O42

Development of a bovine/ovine cytokine
15-plex assay for immunoprofiling of the
cellular response in ruminants

Lesueur J.¹, Barbey S.², Cebron N.¹,
Walachowski S.¹, Lefebvre R.³, Germon P.⁴,
Boichard D.³, Corbiere F.¹, *Foucras G.*¹

¹IHAP, Université de Toulouse, ENVT, INRA,
Toulouse, France, ²UE Le Pin, INRA, Le Pin-aux-
Haras, France, ³GABI, INRA, AgroParisTech,
Université Paris-Saclay, Jouy-en-Josas, France,
⁴ISP, INRA, Université de Tours, Nouzilly, France

Evaluation of the immune status must be
assessed in highly controlled conditions to
be amenable for reproducibility. Furthermore,
whereas the cellular response plays a
major role, immunity has been measured
for a long time through antibody production.
For the purpose of assessing immune cell
responsiveness in ruminants, we developed
and validated a whole blood assay coupled
to a high-throughput multiplex cytokine assay
using the Luminex technology. A custom
Milliplex assay was developed with MERCK-
Millipore company for the measurement of
15 bovine/ovine cytokines of both the innate
and adaptive immunity, and chemokines.
Whole blood samples were collected from
110 dairy cows in an INRA experimental unit,
and immediately stimulated during 24 hours
with heat-killed bacteria (Escherichia coli,
Staphylococcus aureus, and Streptococcus
uberis), Toll-like receptor ligands (FSL-1 for
TLR2, LPS for TLR4, and Gardiquimod for
TLR7-8), or soluble anti-CD3/CD28 monoclonal
antibodies. Cytokine secretion was determined
and compared with a sample prepared in the
same condition but without any stimulant.
Using ANOVA and data mining methods
(PCA, clustering...), we showed that the

been limited compared to humans and mice.
To address this gap, several Immunological
Toolkit initiatives (from 2003 onwards; largely
funded in the UK and USA) aimed to prioritise
new reagent development against research
gaps. Today, all these efforts have produced
thousands of reagents and associated
methods, but a global overview of availability
and applicability is lacking.

After feedback from several national and
international workshops, we have developed
a searchable and highly scalable veterinary
immunology reagent database, accessed
through a website; immunological toolbox.
co.uk. This provides detailed knowledge on
existing reagents, as well as reagents that are
being developed. As such, we aim to highlight
gaps in capability and avoid the duplication of
effort, which itself can provide strong evidence
to support investment in specific research
and development. Information presented will
include cross-reactivity between species
and associated applications alongside a
comments and rating feature to facilitate
continual community scrutiny and improvement.
Frameworksto facilitate collaboration,
exchange or purchase from the owner or
company will also be available.

The website also links directly to the practical
components of the Pirbright/Roslin Toolbox. This
includes the generation of new reagents based
on community priorities, and the capability to
recombinantly express and engineer monoclonal
antibodies. Consequently, this first version of
the website has largely been driven by the
information available from Pirbright and Roslin
antibody repositories. However, the quality
and quantity of future information will rely on
community engagement. Discussions at an
international level will be extremely valuable
in helping to remove barriers and promote
veterinary immunological research.
protocol is able to detect differences between
bacteria, with *S. uberis* being the most potent
to induce a response, compared to *S. aureus*
and *E. coli* that were distinct but more closely
related to each other. Similarly, TLR ligands
could be distinguished, and Gardiquimod
was significantly different from the MyD88-
associated TLR2/4-activating ligands. LPS and
*E. coli* provided very similar response profiles
confirming previous data indicating that a large
part of the *E. coli* response is mediated through
LPS signaling. A high variability of response
was detected amongst cattle suggesting
environmental and genetic variations of the
cytokine response. Altogether, results indicate
that cytokine profiling in cattle is achievable
and can be used in further work to delineate
more precisely the responsiveness of cattle in
various situations, including variability of the
genetic background.

O43

CD40-targeted immunization as a method
for rapid antiserum production and epitope
mapping

Vuong C.N.¹, Chou W.-K.², Hargis B.M.¹, Bielke L.R.³, Berghman L.R.²

¹University of Arkansas, Poultry Science,
Fayetteville, United States, ²Texas A&M
University, Poultry Science, College Station,
United States, ³Ohio State University, Animal
Science, Columbus, United States

The CD40 receptor is expressed on all
professional antigen presenting cells and
is integral to the activation and subsequent
production/affinity-maturation of antibodies.

Previously published data from our laboratories
demonstrated that targeting an antigen to
chicken CD40 receptor induced more rapid
and robust antibody production specific against
the antigen. Using this method, a panel of
oligopeptides of varying lengths ranging from
9 to 23 amino acids were designed to span
an entire target toxin were synthesized for
the purpose of epitope mapping. The target,
Clostridium perfringens alpha toxin, possesses
two enzymatic functions: hemolytic activity and
phospholipase C activity. By loading this panel
of peptides onto the CD40-targeting platform
and immunizing chickens by subcutaneous
injection (equivalent to 0.33ug of peptide
antigen dosage per chicken), a panel of high
titer peptide-specific antiserum samples was
produced in a week. Peptide-specific antibody
responses measured a minimum mean of
4.6-fold higher than pre-immune baseline,
and as high as 13-fold higher. These serum
samples were used to screen for the ability
to neutralize the alpha toxin's enzymatic
functions. Results demonstrated multiple
candidates for hemolytic neutralization,
but not against phospholipase C. With this
successful proof-of-concept, the CD40-targeted
immunization method was able to produce
functional antiserum for in vitro laboratory
usage a week after a single immunization. This
targeted method is more advantageous than
the traditional style of antiserum production,
which requires multiple immunizations over
a span of several weeks, necessitating long-
term animal care/housing and consumption
of more antigen. Furthermore, because this
platform uses chickens as a host, there is
an increased chance of response against
conserved mammalian domains. Additionally,
this method of rapid antiserum production is
a useful technique for cheaper, faster epitope
mapping and has since been successfully used
for candidate selection in development of a
necrotic enteritis vaccine for poultry.