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