Download Cliptox increased the protection levels induced by an FMDV inactivated vaccine

Adjuvant effect of CliptoxTM on the Immune response induced by an
inactivated vaccine against foot and mouth disease virus in mice
Batista A1., Quattrocchi V 3, Olivera V2.; Langellotti C3;.; Pappalardo, S 2,3.; Mongini C3. Portuondo, D1. and Zamorano, P2,3
1
Toxicology and Biomedicine Center (TOXIMED), Santiago de Cuba, 2Virology Institute. INTA Castelar,Argentina, 3CONICET, Argentina
INTRODUCTION
Foot and Mouth disease (FMD) is an acute, highly contagious viral disease. It is economically important because of the international restrictions
it imposes to cattle commercialization. Routine vaccinations in enzoonotic (non-FMD-free) regions can reduce production impact of the disease.
Mostly available FMD vaccines are inactivated whole-virus preparations which contain oil emulsions as an adjuvant to improve their efficacy.
The effective formulation of FMD inactivated vaccines requires adjuvants and Al(OH)3/saponin and mineral oil-based formulations have been
widely employed in experimental studies. However, some currently available FMD vaccines for pigs have been reported to induce poor immune
responses in swine.
Microparticles have already been shown to be effective delivery systems for vaccine formulation inducing potent cellular and humoral immune
responses. Furthermore, they can protect the antigens against the aggressive conditions such as the low pH, the biliary salts and enzymes.
Cliptox™ is a zeolite. The Zeolites are microparticles playing an important role in immune system regulation. It was reported that silica, silicates,
and aluminosilicates act as non-specific immunostimulators similarly to superantigens with the ability to activate a relatively large fraction (520%) of the T cell population, as well as humoral immunity.
Earlier results obtained in our lab have demonstrated the adjuvant effect of natural microparticles of clinoptilolite (Cliptox™) using two classic T
dependent antigens (ovoalbumin and sheep red blood cells). Mice injected in two subcutaneously doses elicited high titters of specific antibodies
and irrelevant side effects in the site of inoculation.
Adult Balb/C mouse is not susceptible of natural infection with FMDV O1, but it can be experimentally infected by ip inoculation. After
inoculation, the virus replicates in pancreatic cells and the viremia last for 72 hours without clinical symptoms. When the neutralizing antibodies
increase, the viremia ends.
The objective of this study was to evaluate the efficacy of Cliptox™ as adjuvant using inactivated FMDV, to induce protection against FMDV
O1C in our murine model.
The immune response after inoculation of vaccines formulations were studied in the murine model, in order to know the mechanisms involved in
protection.
MATERIALS AND METHODS
Improvement of the protection using Cliptox in the vaccine
Group
% protection
Protected / challenged
FMDV
20
2/10
Cliptox +FMDV
90
9/10
PBS or Cliptox
alone
0
0/4
Protection levels induced by formulations containing 0.5ug FMDV: FMDV plus
Cliptox versus percentage of proteccion induced by 0.5ug FMDV or adjuvant alone
and non vaccinated control (PBS).
The level of Ig A against FMDV in mucose is increased by
the use of Cliptox-FMDV vaccine (23 dpv)
Animals: adult mice BALB/c. All the experiments were performed under the international rules of animal welfare.
Virus: inactivated FMDV strain O1Campos was used for vaccine formulations and ELISA tests.
Viral challenge was performed with infectious O1Campos virus (under biosecurity regulations at Biosecurity laboratories NSB 3A of SENASA).
Vaccines: were formulated using 1 ug/doses of Cliptox and 0.5 ug/doses of Virus.
Immunizations and infections: mice were immunized sc at 0 and 10 days (group x2) or only at 0 day with 0.2 ml of each formulation; control
animals were inoculated with saline solution (N group). For mice challenge, mice were infected with 0.5 ml of FMDV (104,5 DICT50/ml) ip.
Viral challenge in murine model: At 23 dpv, mice were infected and bled 24 hours later, heparinized blood was spread in BHK-21 cells. It was
considered that the animal was not protected if the cell layer possessed cytopathic effect and protected, if the cell layer did not present
cytopathic effect after a blind passage. Animals inoculated with PBS were included as positive infection controls, which turned out to be
infected in every viral challenge Antibodies against FMDV measurement: the total specific antibodies in murine serum were determined by
Liquid Phase ELISA. Ab titers are expressed as the negative logaritm of the highest dilution of serum that inhibits color development in more
than 50% of the average value obtained in absence of serum.
The isotypes were determined with Sandwich ELISA.
IgA against FMDV in saliva were detected by sandwich ELISA.
Murine cells obtention and staining: mice were euthanised, the spleen was removed. The cells were marked with fluorescent mAbs from ebioscience: CD11c+/MHCII+ (Dendritic cells), F480+ (macrophages) and were analized by flow cytometry.
2.5
2
OD
1.5
1
0.5
0
Cliptox+FMDV
FMDV
Negative Control
Groups
RESULTS
The number of Dendritic cells and Macrophages are increased
with the use of Cliptox-FMDV vaccine
A
The formulation Cliptox-FMDV is non toxic
B
A
Membrane corioallantoic of
chicken`s embryo alter the
aplication of
A-Negative
control
or
Cliptox-FMDV
B-Positive Control.
% Total CD11c and Class II positive Cells
0.8
0.7
0.6
FMDV antibody levels induced by vaccines
0.5
in the murine model.
0.3
0.4
0.2
0.1
0
3
Cliptox+FMDV
B
FMDV
(FMDV)x2
1.5
Cliptox+FMDV
1
(Cliptox+FMDV)x2
0.5
0
0
5
10
15
20
25
dpv
Total Ab induced at different days post vaccination (liquid-phase ELISA test).
Each point represent the mean of Ab levels per group of animals.
% F4/80+ Mac3+ Cells in Gate 2
Ab titre
Negative Control
Negative Control
2
12
10
8
6
4
2
0
Cliptox+FMDV
FMDV
Negative Control
Group
FMDV isotype antibody levels induced by vaccines
Dendritic cells A- and Macrophages B- from spleen were
measured at 2 dpv, the results are expressed as total numbers of
positive cells in the analized gate.
in the murine model (23 dpv).
CONCLUSIONS
3,5
IgG1
3
A b s o rb a n c e 4 0 5 n m
FMDV
Group
2.5
IgG2a
2,5
IgG2b
IgG3
2
1,5
1
0,5
0
Negative Control
FMDV
Cliptox+FMDV
(FMDV)x2
(Cliptox+FMDV)x2
Groups
Isotype profile in sera taken from mice at 23 days post vaccination. The OD
from each animal sera was measured and then the mean was calculated.
In this study we evaluated the capacity of induce specific and protective immune response against
FMDV
We demostrate that Cliptox, a mineral microparticle, is non toxic with adjuvant activity.
The formulation Cliptox-iFMDV increased the specific antibodies levels and the the protection against
the virus in the murine model.
The different isotype profile elicited by Cliptox-iFMDV indicate a Th1/Th2 response.
Since it is generally considered that good mucosal immunity will contribute to protection against
infection with FMDV, the induction of mucosal immunity (IgA in saliva) by parental immunization is an
important result.
Since vaccines formulated with Cliptox-iFMDV induce an increase in dendritic cells, macrophages and
monuclear phagocytes in spleen, our hypothesis is that vaccine formulations containing the adjuvant
could promote the presentation of the virus so it could increase the immune response and the
protection.
All these results demonstrate that Cliptox could be a good candidate for the formulation of a vaccine