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ADJUVANTS
Carolina TafallaAnimal Health Research Center (CISA-INIA)
TYPES OF VACCINES
LIVE, ATTENUATED VACCINES
INACTIVATED VACCINES
RECOMBINANT VACCINES
DNA VACCINES
VIRUS LIKE PARTICLES (VLPs)
Safe
ty
Im
mu
no
gen
icity
-What is an adjuvant?
A substance that we add to a vaccine to increase its immunogenicity
WHY DO WE NEED ADJUVANTS?
-Not needed in live attenuated vaccines, as their replicative capacityalready provides a delivery method to the antigen presenting cells.Components of the pathogen act as immunostimulants.
-Especially needed when we use subunit vaccines or when we want toimmunize through mucosal routes
TYPES OF ADJUVANTS
Signal 1 facilitators Signal 2 facilitators
- They include all variants of delivery systems,including depot adjuvants
- They affect antigen residence time, the spatio–temporal behavior of the antigen (antigengeography) and the amount of the antigen thateventually reaches the adaptive immune cellreceptors
- Examples: oil emulsions
- Do not directly affect theconcentration and distribution ofantigen between injection site anddraining lymph nodes over time
- They provide co-stimulatory signalsduring the antigen recognition phase,thus increasing the immune responseor skewing it to provide the mostsuitable immune environment for theestablishment of protection
- Examples: ligands of innatereceptors, cytokines
OIL-BASED EMULSIONS
generate long-term immune responses
Low HLB value High HLB value Intermediate HLB value
well tolerated but induce ashorter term immune response
W/O O/W W/O/W
Emulsion= dispered phase + continuos phase + surfactantHLB= hydrophilic: lipohilic balance
long-term immune responses, but strong side effect
SIGNAL 1 FACILITATORS
Freund´s complete adjuvant-W/O. Heat-killed Mycobacteria and a mineral oil with surfactant-Strong side effects-Not always worked in fish
Freund´s incomplete adjuvant-Lacks the mycobacterial components of the emulsion, being therefore just a W/Oemulsion-Less side effects, but still some like peritonitis-Evidences of good effects in fish
Montanide-Based on either mineral oil, non-mineral oil or a mixture of both-May be used to manufacture different type of emulsions, W/O, O/W or W/O/W, for use inboth mammals and fish-Less side effects-Evidences of good effects in fish
Other mineral oils-AJ-oil (Alphaject 5200) used in some vaccines commercialised by Pharmaq
MONTANIDE™ in the fieldStreptococcusAeronomasVibriosisYersiniaPasteurellosisFuronculosis
SIGNAL 1 FACILITATORS
- Work resonably well for antibacterial vaccines delivered intraperitoneally
- Not effective for viral or parasitic diseases
- Strong side effects
Noia et al. FSI 2014; 38: 244-254.
C. Secombes
SIGNAL 2 FACILITATORS
- provide co-stimulatory signals during the antigen recognition phase
- Skewing the immune response to provide the most suitable immune environment forthe establishment of protection
SIGNAL 2 FACILITATORS
-Aluminium salts
Some of the few adjuvants that have been allowed and considered safe to use in human vaccines
Induce Th2 responses
Activates NLRP3 inflammasome and DCs
Only a few studies have used aluminium adjuvants in the optimization of fish vaccines
-TLR ligands
-Cytokines
Chemokines, pro-inflammatory cytokines, IFN-related cytokines
SIGNAL 2 FACILITATORS
Poly I:C
Lipopeptides
Flagellin
CpGs
LPS
b-glucans
Saponins
ISCOMs (cholesterol,phospholipid andsaponin)
Enterotoxins
SIGNAL 2 FACILITATORS
SEARCH FOR EFFECTIVE ADJUVANTS FOR DNA
VACCINES IN FISH
DNA VACCINES AGAINST FISH RHABDOVIRUS
i.m. injection0.01-10 mg
CHEMOKINES AS ADJUVANTS
CHEMOKINES
Attract immune cells to inflammation siteRegule immune function of recriuted cells
Conditionate the specific immune response
100 ml PBSI.M.
100 ml PBS1 mg pcDNA
I.M.
100 ml PBS1 mg pCK5B
I.M.
100 ml PBS1 mg pCK6
I.M.
100 ml PBS1 mg pCK7A
I.M.
Head kidney and spleen removed for RNA extraction at days 1, 2, 5 and 7
Evaluation through real-time PCR of the levels of expression of different immune genes
VERIFICATION OF CHEMOKINE EXPRESSION IN THE MUSCLE
C pcDNA pCHEMOKINE
Experimental design
Head Kidney
Spleen
IL1-β
0,0
5,0
10,0
15,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
TNF-α
0,0
5,0
10,0
15,0
20,0
25,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
IL1-β
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
TNF-α
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A* * * *
*
** *
*
*
*
*
*
Levels of expression of pro-inflammatory genes
Head Kidney
Spleen
IFN2
0,0
1,0
2,0
3,0
4,0
5,0
6,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
Mx
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
IFN-γ
0
5
10
15
20
25
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
IFN2
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
Mx
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
Day 1 Day 2 Day 5 Day 7
Days post-injectionR
ela
tiv
e e
xp
res
sio
n
pcDNA
pCK5B
PCK6
PCK7A
IFN-γ
0,0
5,0
10,0
15,0
20,0
25,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
PCK6
PCK7A
*
*
***
* **
*
*
**
*
*
*
*
*
*
*** **
*
* *
*
Levels of expression of interferon-related genes
Head Kidney
Spleen
CD4
0,0
0,5
1,0
1,5
2,0
2,5
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
* ***
CD8
0,0
1,0
2,0
3,0
4,0
5,0
6,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A* *
CD4
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
* *
** * *
CD8
0,0
1,0
2,0
3,0
4,0
5,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
*
*
**
*
**
*
MHC-II
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A*
*
**
***
*
MHC-II
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
Day 1 Day 2 Day 5 Day 7
Days post-injection
Re
lati
ve
ex
pre
ss
ion
pcDNA
pCK5B
pCK6
pCK7A
**
*
*
Levels of expression of “marker” genes
Adjuvant effect of pCK6 in DNA vaccination
Despite these immunostimulatory effects co-injection of pCK6 with a VHSV DNA vaccinedid not increase the protection conferred.
% m
orta
lity
Treatment
Non-vaccinated
Vaccinated pVHSV
pVHSV + pCK6 (5 ng)
pVHSV + pCK6 (50 ng)
- 12 g rainbow trout- Plasmids were encapsulated in alginate- Feed to the fish 1 week, 1 week out, 1 week- Challenge with VHSV after 6 weeks
Experimental design
-Plasmids coding for VHSV G protein and CK5B, CK9 and CK12 chemokines
Antigen Delivery % Mean accumulated
mortality Alginate/pBI Feed 91Plasmid pBI Intramuscular
injection91
Plasmid pcDNA3-vhsG Intramuscular injection
0
Plasmid pBI-vhsG Intramuscular injection
9
Alginate/pBI-vhsG Feed 78Alginate/pBI-vhsG-CK5/9/12 Feed 81
Alginate/pBI-vhsG Intubation 80Alginate/pBI-vhsG-CK5/9/12 Intubation 92
Results
CpGs AS ADJUVANTS
Introduction of a multi-copy CpG fragment in a VHSV DNA vaccine
Xho I Sal I
Xho I
Xho IXho I
Xho ISal I
1 CpGXho I
pVHSV-2CpG
pVHSV-4CpG
pVHSV
vaccine
CpG-rich fragment
3CpG
gtcgac-accgatgtcgttgccggtgacg tccatgtcgttcctgatgct tcgtcgttggttgtcgttttggt
tcgtcgttttgacgttttgtcgtt ttcgtcgttttgtcgttttgtcgt ttcgtcgttttgtcgttttgtcgtt
tccatgacgttcctgacgtt accgataacgttgccggtgacg tccatgacgttcctgatgct
tccatgacgtccctgatgct ctcgag
Sal I1681 1669 2113
2102 2143 2006
1826 1670 1668
1651Xho I
gtcgac-accgatgtcgttgccggtgacg tccatgtcgttcctgatgct tcgtcgttggttgtcgttttggt
tcgtcgttttgacgttttgtcgtt ttcgtcgttttgtcgttttgtcgt ttcgtcgttttgtcgttttgtcgtt
tccatgacgttcctgacgtt accgataacgttgccggtgacg tccatgacgttcctgatgct
tccatgacgtccctgatgct ctcgag
Sal I1681 1669 2113
2102 2143 2006
1826 1670 1668
1651Xho I
GROUP 1100 ul PBS
intramuscular
GROUP 2100 ul PBS
+ 1 ug pcDNAintramuscular
GROUP 3100 ul PBS
+ 1 ug pVHSVintramuscular
GROUP 4100 ul PBS
+ 1 ug pVHSV-2CpGintramuscular
GROUP 5100 ul PBS
+ 1 ug pVHSV-4CpG intramuscular
Days 2, 7 and 14 p.v.
RNA extraction to study gene
transcription
Serum
Neutralizingcapacity
Day 30 p.v.
Experimental design of the vaccination experiment
Spleen and muscle
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
Muscle
Spleen
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpG pVHSV-4 CpG0
2
4
6
8
200
400
Fo
ld o
f in
cre
ase
Day 2 Day 7 Day 14
a
Day 2 Day 7 Day 14
a, b aa
a, b
a
a, b
a, b
a
a
a
1. Control2. pcDNA3. pVHSV4. pVHSV-2CpG5. pVHSV-4Cpg
Martinez-Alonso et al. Vaccine 2011; 29: 1289-1296.
Mx transcription levels after vaccination
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1. Control2. pcDNA3. pVHSV4. pVHSV-2CpG5. pVHSV-4CpG
Spleen
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
1,5
3,0
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
1,5
3,0
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
1,5
3,0
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
1,5
3,0
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
1,5
3,0
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
0,2
0,3
0,4
1,5
3,0
Fo
ld o
f in
cre
ase
Muscle
a
a a
a
a, b
a
a, ba, b a, b
Martinez-Alonso et al. Vaccine 2011; 29: 1289-1296.
IFNg transcription levels after vaccination
Day 2 Day 7
Day 7
Day 14
Day 14
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Day 2
1. Control
2. pcDNA3. pVHSV4. pVHSV-2CpG5. pVHSV-4CpG
Muscle
Spleen
PBS Empty pVHSV pVHSV-2 CpGpSV-4 CpG0,00
0,01
0,02
0,03
0,08
0,09
0,10
Fo
ld o
f in
cre
ase
PBS Emp pVHSV pVHSV-2 CpGpVHSV-4 CpG0,00
0,01
0,02
0,03
0,08
0,09
0,10
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,00
0,01
0,02
0,03
0,08
0,09
0,10
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,00
0,01
0,02
0,03
0,08
0,09
0,10
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,00
0,01
0,02
0,03
0,08
0,09
0,10
Fo
ld o
f in
cre
ase
a, b a, b
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,00
0,01
0,02
0,03
0,080,090,10
Fo
ld o
f in
cre
ase
a, b
Martinez-Alonso et al. Vaccine 2011; 29: 1289-1296.
MHC I transcription levels after vaccination
Day 2
Day 2
Day 7
Day 7
Day 14
Day 14
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1. Control2. pcDNA3. pVHSV4. pVHSV-2CpG5. pVHSV-4CpG
Muscle
Spleen
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
1
2
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
1
2
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
1
2
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
1
2
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
1
2
Fo
ld o
f in
cre
ase
PBS Empty pVHSV pVHSV-2 CpGpVHSV-4 CpG0,0
0,1
1
2
Fo
ld o
f in
cre
ase
b a
a a
a, b
a
b b
a
Martinez-Alonso et al. Vaccine 2011; 29: 1289-1296.
IL-1b transcription levels after vaccination
Day 2
Day 2
Day 7
Day 7
Day 14
Day 14
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
0
25
50
75
100
10 50 250
Inverse of trout serum dilution
Inh
ibit
ion
of
VH
SV
in
fecti
vit
y, %
of
co
ntr
ol
pcDNA
pVHSV
pVHSV-2CpG
pVHSV-4CpG
Martinez-Alonso et al. Vaccine 2011; 29: 1289-1296.
Serum neutralizing activity after vaccination
Inverse of trout serum dilution
Inh
ibit
ion
of
VH
SV in
fect
ivit
y, %
of
con
tro
l
USE OF RECOMBINANT IL6 AS AN ADJUVANT FOR AN IPNV
INACTIVATED VACCINE
GROUP 1100 ul PBS
intraperitoneal
GROUP 2100 ul PBS
+ 100 ng IL6intraperitoneal
GROUP 3100 ul PBS
+ 2x109 TCID50 IPNVintraperitoneal
GROUP 4100 ul PBS
+100 ng IL6+2x109 TCID50 IPNV
intraperitoneal
Days 2, 6 and 15 p.v.
ELISPOT and levelsof MHC II
Serum
Antibody titers
Experimental design of the vaccination experiment
Days 2, 6, 15 and 30 p.v.
Spleen and kidney cells
0
100
200
300
400
500
600
700
800
Spo
t Fo
rmin
gC
ells PBS
IL6
IPNV
IPNV IL6*
*
*
0
500
1000
1500
2000
MH
C-I
I MFI PBS
IL6
IPNV
IPNV IL6
* * *
Day 2 Day 6 Day 15
Day 2 Day 6 Day 15
Effects of IL6 on spleen IgM+ B cells after vaccination
The addition of IL6 to the vaccinesignificantly increases the numberof IgM-secreting cells in thespleen
The addition of IL6 to the vaccinesignificantly decreases theamount of surface MHC II onsplenic IgM B cells
0
100
200
300
400
500
600
Spo
t Fo
rmin
gC
ells PBS
IL6
IPNV
IPNV IL6*
* *
0
500
1000
1500
2000
2500
3000
3500
4000
MH
C-I
I MFI
PBS
IL6
IPNV
IPNV IL6*
Day 2 Day 6 Day 15
Day 2 Day 6 Day 15
Effects of IL6 on kidney IgM+ B cells after vaccination
The addition of IL6 to thevaccine significantly increasesthe number of IgM-secretingcells in the kidney
The addition of IL6 to thevaccine significantly decreasesthe amount of surface MHC IIon kidney IgM B cells
0
0.1
0.2
0.3
Control IPNV IPNV + IL6
*
*
*
0
0.1
0.2
0.3
Control IPNV IPNV + IL6
Day 2 Day 6
Anti-IPNV Abs
0
0.1
0.2
0.3
Control IPNV IPNV + IL6
**
0
0.1
0.2
0.3
Control IPNV IPNV + IL6
*
Day 15Day 30
Effects of IL6 on serum IPNV-binding IgM titers
OTHER PROMISING SIGNAL 2 ADJUVANTS FOR USE IN FISH
b-GLUCANS AS ADJUVANTS
0
0.0005
0.001
0.0015
0.002
Co conc. 1 conc. 2 conc. 3 conc. 4
TNF
0
0.002
0.004
0.006
0.008
Co conc. 1 conc. 2 conc. 3 conc. 4
IL-1
0
0.001
0.002
0.003
Co conc. 1 conc. 2 conc. 3 conc. 4
IL-8
0 10 50 100 200
0 10 50 100 200
0 10 50 100 200
Rel
ativ
eex
pre
ssio
nR
elat
ive
exp
ress
ion
Rel
ativ
eex
pre
ssio
n
RTS11 stimulated with different glucan doses
b-GLUCANS AS ADJUVANTS IN FISH
Pvana et al. 2016. Braz J Med Biol Res 25; 49(8)
Silver catfish; BSA as model antigen; ip vaccination
Diao et al. 2013. Vet Immunol Immunopathol 156: 167-175
Flounder; E. tarda recombinant; im vaccination
FLAGELLIN AS ADJUVANT
FLAGELLIN AS ADJUVANT IN FISH
Liu et al. 2017. Vaccine 35(2):369-374.
Turbot; Formalin-killed cells (FKC); ip injection.
Jia et al. 2013; Fish Shellfish Immunol 34: 514-520
Flounder; subunit vaccine V. anguillarum; ip injection.
CONCLUSIONS
- In fish, signal 1 adjuvants work efficiently for intraperitoneally deliveredantibacterial vaccines although strong local side effects are elicited
- New adjuvants should be developed for antiviral and antiparasiticvaccines, as well as for vaccines delivered through alternativeadministration routes
- Chemokines do not seem as adequate adjuvants for fish DNA vaccinesagainst rhabdovirus
- The introduction of instrinsic CpG motifs in DNA vaccines appears aspromising strategy to increase their immunogenicity
- Recombinant IL6 increases the immunogenicity of an inactivated virus
b-glucans and flagellin also seems as promising adjuvants for use inaquaculture.
THANK YOU FOR YOUR ATTENTION
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