EuFMD Open Session

1. ASSESSMENT OF POTENCY AND EFFECTIVENESS OF HEPTA-VALENT FMD VACCINE OIL
ADJUVANTED (ISA 206) IN EGYPT
Abdel-Hamid Bazid1, Haitham M. Amer2, S.E Mahdy3, Mohamed A. Nayel4, M. Attia5, Nada Maklad 1,5, Momtaz Wasfy5, Momtaz
Shaheen6 and Magdy M. El-Sayed5,7
Introduction
Foot and mouth disease (FMD) is a terrible highly contagious viral disease affecting most domestic animals, leading to severe economic
impacts. Vaccination is the most effective strategy for controlling the infection.
Material and methods
An inactivated hepta-valent FMD oil adjuvanted vaccine was prepared from the following virus strains: A-Iran 05, A-Africa(GIV), O-
PanAsia2, O- Manisa69, OEA-3, SAT-2 Gharbia 12, and SAT-2 Libya 18. The vaccine potency was evaluated in three groups (five calves each)
of 6-8-month-old, and 50 adult dairy cattle under field conditions. All were vaccinated with 3 ml of the prepared vaccine and groups of the
young calves were challenged after 28 days by the inoculation of 104 MLD50 of one of the wild-type viruses of serotypes A, O, or SAT-2
strains via the intra-dermo-lingual route. Mock groups (two calves each) served as negative non-vaccinated controls during the challenge
test Adult dairy cattle were assessed for seroconversion using the virus neutralization test (VNT) after 30 days.
Results
All calves displayed complete protection against the challenge with the different serotypes of FMD virus (FMDV) as compared to the
control groups. Serum samples collected after the primary and booster immunizations at 0 and 30 days showed protective levels of
antibodies at 30 days postvaccination [VNT: 1.57±0.16, 1.60±0.2, and 1.52±0.17 against serotypes A, O, and SAT-2, respectively]. Antibodies
persisted till the end of the experiment, with a peak value around 60 days postvaccination [VNT: 1.75±0.18, 1.78±0.17, and 1.76±0.21 for
serotypes A, O, and SAT-2, respectively].
Discussion
The prepared heptavalent FMD vaccine is potent and capable to provide a protective immune response in both experimental and field
conditions.
Abstract
References
Jamal, SM and Belsham, GJ (2013). Foot-and-mouth disease:past, present and future. Vet. Res., 44: 1-14
Brückner, G and Saraiva-Vieira, V (2010). OIE strategy for the control and eradication of foot and mouth disease at regional and global levels. Compendium of technical
itemspresented to the OIE World Assembly of Delegates and to OIE Regional Commissions. PP: 187-211.
Petrovsky, N and Aguilar, JC (2004). Vaccine adjuvants: current state and future trends. Immunol. Cell Biol., 82:488-496.
Park, ME; Lee, SY; Kim, RH; Ko, MK; Lee, KN and Kim, SM (2014). Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs
and goats. Vaccine. 32: 5221-5227
Cloete, M; Dungu, B; Van Staden, L; Ismail-Cassim, N and Vosloo, W (2008). Evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the SAT
serotypes. Onderstepoort J. Vet. Res., 75: 17-31.
OIE (2012). Foot and mouth disease. In: Manual of diagnostic tests and vaccines for terrestrial animals. OIE, Paris,France.
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.05_FMD.pdf., Chapter 2.1.5.
Results
FMD is an acute and highly contagious viral disease of cloven-hoofed animals. It belongs to the genus Aphtovirus of the Picornaviridae
family which includes seven distinct serologically serotypes and many (sub)lineages (Jamal et al., 2013). One of the most important
strategies for controlling and eradicating FMD is vaccination with high quality vaccines, especially in enzootic areas (Brückner et al., 2010).
The goal of vaccination is to generate immune responses aganist the administered antigen which should provide long-term and strong
protection against infection. The most important factors that affect the immunogenicity of FMD vaccines is the selection of vaccine strains
and the nature of the adjuvant. FMDV serotypes (A,O and SAT-2) are endemic in Egypt, as well as many molecular reports describe
circulation of many topotypes, lineages and genotypes within the three serotypes So, updating vaccine seeds with the predominantly
circulating local strains is important for ensuring optimum protection. The induction of strong and long-lasting immune responses with
inactivated vaccines often requires the addition of a potent and safe adjuvant (Petrovsky et al., 2004). Many types of adjuvants are
employed in veterinary vaccines, however, alum-based, and mineral oil-based adjuvants with or without saponin are most frequently used
for inactivated FMD vaccines (Park et al., 2014). Unlike alum-based adjuvant vaccines, oil-based adjuvant vaccines can overcome
interference by maternal antibodies in neonates and can consequently be applied earlier in life (Iyer et al., 2000). Montanide ISA 206 VG is
a mineral oil-based adjuvant developed by SEPPIC. According to the manufacturer, Montanide ISA 206 VG water-in-oil-in water (W/O/W)
emulsion is robust, stable, easy to inject, induces strong and long-lasting protection and is especially suitable for antigens with a relatively
low immunogenicity (SEPPIC, 2010). In the case of FMD, the serum neutralization test (SNT) is serotype-specific and is considered highly
sensitive to antibodies against the FMD virus (Selim et al., 2010; OIE, 2012). Potency testing is another reliable method of estimating
vaccine efficacy by determining the protection of cattle vaccinated with different vaccines after being challenged with a homologous virus
(OIE, 2012). In the present study, Potency and effectiveness of hepta-valent FMD oil-based FMD vaccine formulated with Montanide ISA
206 VG W/O/W emulsion adjuvant, with the following virus strains/serotypes (A-Iran05, A-Africa-IV, O-PanAsia2, O-Manisa, O-EA3, SAT-2
Garbia and SAT-2 LIB-12)were evaluated
Introduction
Material and methods
FMD virus strains:
All virus work was conducted in biosafety level 3 laboratories of MEVAC company, Egypt. FMDV serotypes/strains were propagated in BHK-21
suspension cells for preparation of viruses. Aseptically, the harvested FMD viruses were clarified using Millipore filters (Millisak®Pod Deth filter Cat#
MC0HC054H1) to remove cell debris.
Virus inactivation and concentration:
FMD viruses were inactivated by two cycles 3mM binary ethylamine (BEI) (Barteling and Cassim, 2004). The excess of BEI was neutralized using
sterile 6mM sodium thiosulphate. The inactivated antigens were concentrated using TFF filter, the concentrated antigens were eluted with Tris-Kcl buffer
pH 7.6 (Barteling and Meloen, 1974). The 146S particles in the concentrated antigen preparations were estimated by using sucrose density gradient
(Doel and Chong, 1982).
Antigen preparation and vaccine formulation:
The inactivated vaccine consisted of an equal volumes of oil phase (Montanide ISA 206VG, Seppic, France) and aqueous phase which were mixed
thoroughly according to manufacturing instructions.
Safety and sterility test:
Prepared vaccine was tested for viral and bacterial sterility. The vaccine was inoculated into two calves by one dose. Four days later two doses of the
vaccine were inoculated subcutaneously (S/C). The inoculated calves were observed for ten days after inoculation (OIE, 2012).
Challenge test:
Twenty-one native calves aged from 6-8 months were used. These calves were clinically healthy and free from antibodies against FMDV as tested by
SNT. The evaluated vaccine was inoculated S/C into 15 calves at the dose rate of 3 ml /animal. The other six calves were injected by the same route with
the adjuvant only were kept as control. Two samples were taken from all calves, the first one just before vaccination and the second one at 28 days post
vaccination. At 28 days post vaccination all calves are challenged with the virulent FMD homologous strain viruses with titer of (104 MLD50) inoculated
intra-dermolingually (OIE, 2012).
Serum neutralizing antibody assay:
SNT has been carried out for quantitative estimation of neutralizing antibodies against FMDV on the sera collected just before vaccination and at 28
days post vaccination. SNT was performed with BHK-21 in flat-bottomed tissue culture grade microtiter plates. The SNT was performed against the
homologous FMDV strains. The test was performed as described in OIE manual 2012 (OIE, 2012) briefly the collected sera are inactivated at 56ºC for
30 minutes before testing. The collected samples were diluted starting from 1/4 to 1/64 and tested against 100 TCID50 (50% tissue culture infective dose)
of FMDV previously titrated. The titers were calculated and expressed as log10
In conclusion,
1. the results of this study showed that a single administration of ISA 206 VG oil-based adjuvant Heptavalent vaccine induced high mean neutralizing antibody titers at 28 days post vaccination as
well as it induced complete protection in vaccinated animals after challenge by 10 4 MLD50 homologous FMD strain.
2. Under field condition the Heptavalent vaccine provided superior clinical protection after two doses (prime and poster doses) and the protection extended for 4 months according to the presented
results
3. Th results suggest that the vaccine formulated with ISA 206 VG (W/O/W) emulsion oil-based adjuvant can be a good and effective vaccine in enzootic countries such as Egypt.
Days post
injection
Calf 1 Calf 1
Rectal
Temp. (ᵒC)
Injection
site
Rectal
Temp. (ᵒC)
Injection
site
1 38.2 38.3
2 38.5 38.6 Swelling
3 38.5 Swelling 38.6 Swelling
4 38.6 Swelling 38.7 Swelling
5 38.3 Swelling 38.6 Swelling
6 38.4 Swelling 38.4 Swelling
7 38.2 38.3 Swelling
8 38.2 38.3 Swelling
9 38.2 38.2
10 38.2 38.2
Serotype # of
animals
SNT titers
(log10)
Lesion Pro (%)
0 DPV 28DPV Tongu
e
Feet
A-Africa (GIV) 5 0.20 1.8 1/5 0.0 100%
O-EA3 5 0.00 1.7 2/5 0.0 100%
SAT-2 (Lib12) 5 0.00 1.6 3/5 0.0 100%
Control A-Africa
(GIV)
2 0.00 NA 2/2 2/2 0.0%
Control O EA3 2 0.00 NA 2/2 2/2 0.0%
Control SAT-2
Lib-12
2 0.00 NA 2/2 2/2 0.0%
SEPPIC (2010). Montanide ISA 61 VG. In S. Inc (Ed.) abbreviated Montanide ISA 61 VG. http://www.seppic. com/file/galleryelement/pj/7d/7b/48/55/4524-technicalbulletin-mtd-
isa-61-vg3096961497520821468.pdf.
Selim, A; Abouzeid, N; Aggour, A and Sobhy, N (2010). Comparative study for immune efficacy of two different adjuvants bivalent FMD vaccines in sheep. J. Am. Sci., 6:1292-
1298.
Iyer, A; Ghosh, S; Singh, S and Deshmukh, R (2000). Evaluation of three ‘ready to formulate’ oil adjuvants forfoot-and-mouth disease vaccine production. Vaccine. 19:1097-
1105.
Barteling SJ and Cassim NI, 2004. Very fast (and safe) inactivation of foot-and-mouth disease virus and enteroviruses by a combination of binary ethyleneimine and
formaldehyde. Dev Biol, 119: 449-455
Barteling SJ and Meloen RH, 1974. A simple method for the quantification of 140S particles of foot-and-mouth disease virus(FMDV). Arch Gesamte Virusforsch, 45: 362-364.
Doel TR and Chong WK, 1982. Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus. Arch Virol,73: 185-191.
Table (1)In vivo safety of prepared FMD vaccine
Table (2)The challenge test for vaccinated cattle with A-Africa (GIV), O-EA3 and SAT-2 (Lib12)
Figure (1)Mean FMD serum neutralization antibody titers in vaccinated cattle with hepta-valent vaccine.
(1) Department of Virology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City,
Menoufia 32897, Egypt.
(2) Virology Department, Faculty of Veterinary Medicine, Cairo University 12211 Giza, Egypt
(3) Veterinary serum and vaccine research institute, Cairo, Egypt.
(4) Department of Medicine and Infectious diseases. Faculty of Veterinary Medicine, University of Sadat
city, Egypt.
(5) Middle East for Veterinary vaccines, second Industrial Area, El-Salhya El-Gedida, El-sharqia, Egypt.
(6) Department of Virology, Animal Health Research Institute (AHRI), Dokki, Giza, Egypt
(7) Department of internal medicine and infectious diseases, Faculty of Veterinary Medicine, Cairo University,
Egypt.