Montanide™ ISA 7 1 R VG:
A robust and flexible adjuvant formulation
for potent and stable poultry vaccines
Juliette Ben Arous”, Sebastien DevUle”, Svetlana Norkinac, Vladimir Borisov’, Tatiana Rozhdestvenskaia’,
Ivan Rozhdestvenskiy’, Jerome Gaucheron, Laurent Dupuis
a SEPPIC, 22 Terrasse Bellini, Paris La Defense, 92806 Puteaux Cedex, France
b SEPPIC, Knox Plaza, Mind Space, Off Chincholi Bunder Rd. Malad West, Mumbai 400064, India
с NPPAVIVAC, Leningradskaya area, village Gorbunki, 188 505 Saint Petersburg, Russian Federation
Contact: sebastien.deville@airliquide.com
Abstract:
Water-in-mineral oil emulsion adjuvants induce a
strong long-term humoral immune response and
are widely used in inactivated poultry vaccines.
However, evolution and destabilization of vaccine emulsion
formulations can be observed during storage when high
concentration of enzymes are present in antigenic media or in
stressing storage conditions. Resistant formulations are
therefore needed to ensure stability of emulsions even in
destabilizing conditions. Moreover, robust adjuvants that
allow flexible ratio of oil and antigenic media in the vaccine
are also needed for the formulation of multivalent vaccines.
Here we show that the new resistant adjuvant Montanide™
ISA 71R VG can resist to destabilizing antigenic media and
conditions, and can be used at flexible ratio in poultry
vaccines. Using a Newcastle disease model vaccine, we
demonstrate that this new adjuvant is also safe and can
improve vaccine efficacy in poultry.
- Introduction:
Vaccine adjuvants are added to vaccine formulations for
multiple purposes, such as increasing the protection given to
animals by veterinary vaccines, enhancing the protection
duration of the vaccine, reducing the number of injections
needed, reducing the antigen load in the vaccine, or orienting
the immune response conferred by the vaccine. Diverse
adjuvants technologies for veterinary vaccines have been
developed in the last decades [ 1 ].
Water-in-mineral-oil (W/O) emulsion adjuvants are
extensively used for the formulation of inactivated avian
vaccines. These strong adjuvants induce long-term efficacy of
poultry vaccines. They consistofa mix of a specific mineral oil
of injectable grade and a surfactant system to stabilize the
vaccine emulsion. It should be noted that a specific care has
to be given to the quality of surfactants as well as to the
nature of the oil to control safety issues [2].
The Montanide™ range of veterinary adjuvants include
emulsion, micro-emulsion and polymeric adjuvants and
have been used extensively for prophylactic veterinary
vaccination in farm and companion animals [3].
Montanide™ W/O adjuvants for poultry are ready to use
adjuvants that induce long lasting protection in chickens and
other avian species [4,5,6].
Chickens receive multiple vaccinations during their life-time.
Multivalent vaccines containing multiple antigens are
therefore used frequently in the field to limit the number of
injections. These vaccines can induce protection against
diverse strains of the same pathogen, or against multiple
pathogens. Multivalent antigenic media are complex media
that contain multiple biological components that can
influence the stability of vaccine emulsions.
Evolution and destabilization of vaccine emulsions during
storage have indeed been observed and can be linked to high
concentration of enzymes such as lipases and esterases in
antigenic media, and to stressing storage conditions. Robust
formulations are therefore needed to ensure stability of
emulsions even in destabilizing conditions, and with flexible
ratio of oil and antigenic media in the vaccine. These features
are therefore especially important for the formulation of
multivalent vaccines.
Here we show that Montanide™ ISA 71R VG, an innovative
emulsion adjuvant dedicated to poultry vaccines, can be
used at flexible ratios and is able to resist to destabilizing
antigenic media and storage conditions. Moreover, we
demonstrate that this adjuvant allows the formulation of safe
and efficient inactivated vaccines against Newcastle disease.
These results show that the safety and efficacy profiles of this
robust adjuvant are comparable to reference water in oil
adjuvants used in poultry vaccines. - Formulation trials: Methods and Results:
Montanide™ ISA 71 R VG is a water-in-mineral-oil adjuvant
based on a specific mannide oleate based surfactant system
that has been designed to increase vaccines stability, and can
be used at flexible ratios (50 to 70% in the vaccine). In order to
control the robustness of this new adjuvant, we developed a
model destabilizing medium (MDM) enriched in enzymes,
which is comparable to destabilizing poultry antigenic media
in the field.
Montanide™ ISA 71 R VG and a standard water in mineral oil
emulsion adjuvant were thus formulated at 70% with either
buffer solution or with MDM, using the same high shear
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emulsification protocol. lOg of each emulsion were
formulated at high shear using a IKA DT20 tube and a IKA
Ultra Turrax Tube Drive emulsification system. 3 x 3 ml of
each emulsion were placed in 3 different haemolysis 5 ml
tubes for stability studies at 4°C, 20°C and 37°C for 6
months. Vaccine emulsions are considered as stable if there
is no defect or small reversible defects, such as oil releases
smaller than 5% or creamings smaller than 15%.
When buffer solution was used, both resisting adjuvant
Montanide™ ISA 71 R VG and classical W/O adjuvant
allowed the formulation of emulsions that were stable over 30
days, even when stored at 37°C. On the other hand, in
combination with destabilizing antigenic media, whereas
vaccines based on resisting adjuvant showed no stability
default in all temperature conditions, standard formulations
showed breakage of the emulsion at 20°C and 37°C, as short
as 7 days post formulation (Figure 1). Emulsions based on
Montanide™ ISA 71R VG showed no defect 6 months after
formulation.
Stability results 7 days after formulation
Formulations 4°C 2 0 T 37°C
Tween/Span –
Placebo
stable stable stable
Tween/SpanMDM
creaming
20% “
creaminq
20%
phase
separation
5%
ISA 71 R –
Placebo
stable stable stable
ISA 71 RMDM
stable stable stable
Stability results 30 days after formulation
Formulations | 4°C 20″C 37°C
Tween/Span –
Placebo
stable stable
oil release
10%
Tween/SpanMDM
creaming
20%
Phase
separation
1 5%
phase
separation
20%
ISA 71 RPlacebo
ok ok ok
ISA 71 R –
MDM
ok ok ok
Stability results of standard and resisting
formulations after 6 months at 20=C
Tween/Span
placebo
Tween/Span ISA 71R
mode! medium placebo
ISA 71R
model medium
T
Figure 1: Results of stability study of Montanide™ ISA 71R VG and
classical adjuvant formulations with buffer solution (Placebo) or
MDM (model medium), 1 week, 1 month and 6 months post
formulation. - Poultry trials: Methods and Results:
To assess safety and efficacy profiles of the new robust
adjuvant Montanide™ ISA 71R VG in chickens, a vaccine
against Newcastle disease was formulated using this
adjuvant. This trial was performed in the facilities of NPP
AVIVAC, Saint Petersburg, Russia.
a/ Vaccine groups and protocol
Inactivated Newcastle disease virus (NDV, La Sota strain,
produced by NPP Avivac) was formulated either with
reference W/O adjuvant Montanide™ ISA 70 VG (used at
70%), with robust adjuvant Montanide™ ISA 71R VG (used at
60%) or in PBS. All vaccines contained the same amount of
antigen. Formulation with Montanide™ adjuvants was
performed at high shear using a IKA 25T mixer and following
Seppic’s recommendations.