Download Development of a fast release immunomodulated vaccine against FMD virus. Induced immunity

DEVELOPMENT OF AN IMMUNOMODULATED
VACCINE OF RAPID LIBERATION AGAINST FOOT
AND MOUTH DISEASE VIRUS. INDUCED IMMUNITY.
QUATTROCCHI, V2.; Langellotti,C2.; Pappalardo, S1,2.; Olivera, V1.; Di Giacomo, S1.; Mongini C2.;
Waldner C 2. y P. Zamorano 1,2.
1Instituto
de Virología,CICVyA, INTA Castelar
INTRODUCTION:
2CONICET
THE PROTECTION INDUCED BY VACCINATION SEEMS TO BE T INDEPENDENT
% DX5+ cells
Athymic mice
Foot and Mouth disease (FMD) is caused by Foot and Mouth Disease Virus (FMDV) which
Tabla 1: protection levels obtained in athymic Nude
protected/challenged
affects cattle, ovines, pigs and several wild species and it causes important economic loss.
mice after vaccination and challenge.
When an outbreak occurs control measures should be applied which include, besides the
4 dpv
7dpv
3/8 (37,5%) 3/8 (37,5%)
sacrifice of the infected animals, a ring vaccination of the surrounding cattle, with a vaccine FMDVi
The response of SN antibodies and totals was similar in
802-FMDVi 7/8 (87,5%) 7/8 (87,5%)
capable of protecting animals against the disease in a short period of time.
Nude and Balb/C mice. (data not shown)
6/8 (75%)
Few emergency vaccines have successfully induced complete protection in a short period of time 206-FMDVi 7/8 (87,5%)
post vaccination and it has been demonstrated that there is no correlation between the
CHARACTERIZATION, BY FLOW CYTOMETRY, OF CELL POPULATIONS MODULATED BY
neutralizing antibodies induced by the vaccination and the protection, so proposing a possible
VACCINATION
protective role, on a short term basis, of other components of the immune response, besides the A 20
18
16
humoral immunity.
Fig 4: means and standard deviation of A) NK (DX5+ cells)
14
It is well described that the adjuvants play an important role on vaccines. In the present work we
12
B) Macrophages (F4/80+ cells) in peritoneal cavity,
10
have studied the protection and the type of immune response induced by inactivated virus
8
measured by flow cytometry using 3 mice/group, are shown.
6
vaccines and immunomodulators in a murine model, at 4 and 7 days post vaccination (dpv). The
*
(* = p<0.05 regarding group FMDVi)
4
*
vaccine which induced better protection levels in this model, was studied in bovines.
2
*
MATERIALS AND METHODS:
0
Animals: adult mice BALB/c and N/NIH(S) nude strain, bovines and pigs negative to FMDV. 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 Box NSB 3A of INTA or at Biosecurity laboratories NSB
3A of SENASA). Vaccines: vaccines were formulated using adjuvants ESSAI IMS 12802 PR (IMS802) Montanide IMS1313 N PR (IMS1313),
ESSAI IMS D 12801 PR (IMS801), ISA206 (W/O/W), ISA70 and ISA773 (W/O) (Seppic), formulations were made following the manufacturer
instructions. Immunizations and infections: mice were immunized intraperitoneally (ip) with 0.2 ml of each formulation; control animals were
inoculated with saline solution (N group). For mice challenge, animals were infected with 0.5 ml of FMDV (104,5 DICT50/ml) ip. Bovines received 2
ml of vaccine by im route. Pigs were infected with 0,5 ml of infectious FMDV (106 DICT50/ml) by intradermic route. Viral challenge in murine model:
vaccinated 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 experiment
presented. Contact challenge in bovines: pigs were experimentally infected. When presenting disease sympthoms they were housed for 3-4 hours
(with circulation every 15 minutes), with the vaccinated bovines, and control animals. Afterwards, the infected pigs were euthanized and the
control animals were housed in a separate room. The animals were studied at 7 days post challenge. Animals that did not present the typical FMD
vesicles were considered protected. Antibodies against FMDV measurement: the seroneutralizing antibodies were determined by
seroneutralization assay using fix virus method. The total specific antibodies in murine serum were determined by Sandwich ELISA and Liquid
Phase ELISA in bovines. The isotypes were determined with Sandwich ELISA. Murine cells obtention and staining: mice were euthanised,
peritoneal washes were perfomed and the spleen was removed. The cells were marked with fluorescent mAbs and were analized by flow
cytometry. Bovine cells obtention and staining: the heparinized blood was sown over Ficoll-Paque TM plus, in order to isolate mononuclear cells.
The cells were marked with mouse mAb and then with sheep anti-mouse antibodies conjugated with FITC or PE, to be analyzed by flow
cytometry. Cytokine measurement: murine and bovine cells were placed on a culture plate, 24-72 hs later the supernatants were collected and
assayed by sandwich ELISA. In vivo NK depletion: it was performed by ip inoculation with anti-asialo-GM1 antibody, a day before vaccination and
a day before challenge. In vivo macrophages depletion: LipClod was inoculated ip and iv, a day after the vaccination. Depletions were assesed by
flow citometry. Opsonization-phagocytosis assay: dilutions of inactivated sera of vaccinated or normal animals were put in contact with inactivated
FMDV marked with [3H]Uridine. Peritoneal cavity cells from the same animal, were mixed with virus-antibody mixtures and then washed in order to
eliminate not associated-virus with the cell fraction. Cells were harvested and the radioactive label incorporated was determined with a liquid
scintillation counter. Statistical analysis: Statistix program was used. T Test of Student was applied to compare between two treatments. A p value
<0,05 was considered an indicator of significant differences.
2 dpv
FMDVi
B
4 dpv
100
% F4/80+ cells
30
20
10
0
FM DVi
4 dpv
7 dpv
3 ug/d
1 ug/d
B
0,5 ug/d 0,1 ug/d 0,05 ug/d 0,02 ug/d
Vaccines were formulated
with the adjuvants ISA206,
ISA773, ISA70, IMS802,
IMS801 and IMS1313, and
were tested in a preliminar
assay of vaccination and viral
challenge in the murine
model (data not shown). It
was decided to continue the
investigation with the IMS802
adjuvant (802-FMDVi
vaccine) which induced the
highest protection level. Also
ISA206 (206-FMDVi vaccine)
was studied, which has
already been used in
emergency vaccines
formulation.
Fig. 2: protection levels
in BALB/c mice
(protected/ challenged
Protection induced at 7 dpv in Balb/C mice
Protection induced at 4 dpv in Balb/C mice
B
A
animals x 100), at A) 4
110
110
100
100
dpv B) 7 dpv. The bars
90
90
80
80
represent the mean of
70
70
60
60
two experiments with 8
50
50
40
40
animals per group,
30
30
20
20
except on groups 802
10
10
0
0
and 206, in which n = 4.
FMDVi
802-FMDVi
802
206-FMDVi
206
FMDVi
802-FMDVi
802
206-FMDVi
206
A N group (2 control
vaccine
vaccine
animals inoculated with
PBS) was included,
THE LEVELS OF SPECIFIC TOTAL Ab INDUCED BY VACCINATION ARE HIGHER THAN
which always had 0% of
THOSE INDUCED BY FMDVi
protection
(data
Fig. 3: total
Abnot
induced
shown).
at A) 4 dpv and B) 7
A
Total antibodies induced at 4 dpv in
Total antibodies induced at 7 dpv in
B
BALB/C mice
BALB/C mice
dpv in vaccinated mice.
0,6
0,8
O.D. of each sera,
*
*
0,7
0,5
0,6
(1/10
dilution)
are
0,4
*
*
0,5
shown. Black line =
0,3
0,4
0,3
0,2
mean O.D. of each
0,2
0,1
group (n=8). Dotted
0,1
0
0
line = cutting edge.
FMDVi
802-FMDVi
206-FMDVi
FMDVi
802-FMDVi
206-FMDVi
0
1
2
3
4
0
1
2
3
4
(*=p<0.05
when
Vaccine
Vaccine
compared with FMDVi
group).different (p>0.05)
SN Ab induced in groups 802-FMDVi and 206-FMDVi were not significantly
from the obtained in group FMDVi, not even at 4 or 7 dpv (data not shown).
Both IgM and IgG (IgG1, 2a, 2b and IgG3) isotypes were found.
Igs anti VFA (O.D. 492 nm)
% P ro t e c t io n
PROTECTION INDUCED BY VACCINATION IN THE MURINE
MODEL.
% Pro t e c t io n
206-FM DVi
Vaccine
NK CELLS ARE NOT PLAYING A KEY ROLE IN PROTECTION
Tabla 2: protection levels in viral
challenge obtained in vaccinated and
4 dpv
7 dpv
NK-depleted or mock-depleted BALB/c
mock- depleted NK-Depleted mock- depleted NK-Depleted mice, at 4 and 7 dpv. The protection
levels are indicated in brackets. The
FMDVi
3/8 (37,5%)
1/4 (25%)
2/8 (25%)
0/4 (0%)
802-FMDVi
8/8 (100%)
3/4 (75%)
8/8 (100%)
3/4 (75%) depletion was ≥ 80%, both in peritoneal
206-FMDVi
7/8 (87,5%)
4/4 (100%)
4/8 (50%)
2/4 (50%) cavity and spleen (determined by flow
MØ PLAY A KEY ROLE IN PROTECTION cytometry).
INDUCED BY VACCINATION
Mice protected/challenged
Mice protected/challenged
Fig. 1: protection levels induced in BALB/c mice vaccinated with
different concentrations of inactive FMDV in PBS. 8 mice per group
were inoculated and challenged at A) 4 and B) 7 dpv. The dose of
inactivated virus selected was the one that induced a low protection
level, in order to be able to differentiate the effect of the adjuvants
used in this work.
Igs anti VFA (O.D. 492 nm)
802-FM DVi
N
FMDVi
802-FMDVi
206-FMDVi
Mock-depleted MØØ-depleted
1/8 (12,5%)
0/8 (0%)
7/8 (87,5%)
1/8 (12,5%)
6/8 (75%)
2/8 (25%)
802-FMDVi VACCINE INCREASED PROTECTION IN
CATTLE
Vaccines formulated with 20ug FMDVi/dose
100
90
80
70
60
50
40
30
20
10
0
5/5
5/5
4/5
3/5
3/5
1/3
1/5
4dpv
7dpv
14dpv
4dpv
802-FM DVi
7dpv
1313-FM DVi
Tabla 3: protection levels in viral challenge induced in
vaccinated MØ-depleted or mock-depleted BALB/c
mice, at 7 dpv. Protection percentages are indicated in
brackets. Depletion was ≥ 90%, in peritoneal cavity and
≥ 60% in spleen. The peritoneal cells from mice
vaccinated
with
802-FMDVi
and
206-FMDV,
incorporated significantly (p<0,05) more marked virus
than the ones from control mice or from FMDVi group, in
the opsono-phagocytosis assays (data not shown).
7dpv
7dpv
FM DVi
COM
Vaccine and days post vaccination evaluated
Fig. 5: protection percentages reached by cattle
vaccinated with 802-FMDVi at 4, 7 or 14 dpv, 1313FMDVi at 4 or 7 dpv, commercial vaccine at 7 dpv or
FMDVi at 7 dpv. The numbers over the bars represent:
number of protected animals/challenged animals.
There was no relationship between animals with high
titers of SN or total specific ab against FMDV (measured
by FL ELISA) and protection in viral chalenge.
VACCINATION WITH 802-FMDVi,
INCREASES MØ POPULATION IN CATTLE
CD14+ peripheral blood mononuclear cells
4000
3500
3000
2500
2000
1500
1000
500
0
3597
3607
802-FMDVi
T0
4 dpv
VACCINATION WITH 802-FMDVi, INCREASES
IFNγγ IN VITRO SECRETION.
T0 4 dpv 7 dpv
IFNγ in vitro secretion by peripheral
Fig. 7: IFNγ
Fig. 6:
blood mononuclear cells
secreted in
0,5
Total CD14+ 0,45
vitro by
0,4
mononuclear 0,35
mononuclear
0,3
peripheral
0,25
peripheral
0,2
blood cells
0,15
blood cells
0,1
(monocytes/ 0,05
from
0
MØ).
3597
3607
3598
3608
3605
vaccinated
802-FMDVi
FMDVi
Normal
bovines
O.D. 492 nm
0,5 ug/d 0,1 ug/d 0,05 ug/d 0,02 ug/d
An increase on the in vitro secretion of IL6, TNFα and IL10
was observed, by cells from the peritoneal cavity or spleen,
of vaccinated mice regarding group N, and an increase of
IFNγ secretion by spleen cells from animals vaccinated with
802-FMDVi or 206-FMDVi regarding vaccinated animals with
VFAi (data not shown).
*
50
40
% P ro te c tio n
1 ug/d
Protection percentages at 7 dpv
%
110
100
90
80
70
60
50
40
30
20
10
0
*
60
ce ll n u m b e r
3 ug/d
A
Vaccine
70
RESULTS
Protection percentages at 4 dpv
In peritoneal cavity and spleen 802-FMDVi and 206-FMDVi
induced a significant increase of neutrophils (p<0,05)
regarding FMDVi. (data not shown).
206-FMDVi
N
*
90
80
SELECTION OF THE ANTIGEN DOSE
%
110
100
90
80
70
60
50
40
30
20
10
0
802-FMDVi
7 dpv
7 dpv
3608
3698
FMDVi
3605
normal
animal number and vaccine
DISCUSSION:
- In the murine model vaccines 802-FMDVi and 206-FMDVi increase the protection levels
compared to FMDVi group, at 4 and 7 dpv in the absence of SN Ab that could be considered as
protective. Protection was related to total specific Ab. In cattle the vaccine protected all animals
at 4 and 7 dpv, in the absence of SN ab that could be considered as protective. This formulation
could be use as an emergency vaccine.
- In mice, protection and humoral immune response were T independent. IgG1, IgG2a and IgG2b
were induced at early times post vaccination, thus indicating a possible stimulation of marginal
zone B lymphocytes and/or B1.
-In the murine model it was demonstrated that MØ are indispensable for protection. On the other
hand NK cells are not essential though they were modulated by vaccination. Antibodies
opsonization and virus-Ab complex phagocytosis by MØ, play an important role in protection. In
cattle, MØ and IFNγ levels were increased by 802-FMDVi, thus indicating that MØ could play a
key role in the immune response induced by this vaccine, in this specie.
- Taken together, these results could explain why vaccines wich induce a low, non neutralizing
Ab levels are capable of protecting animals from viral challenge, at early times post vaccination.