Alum adjuvants :
discovering their hidden
secrets
Ana Carolina Pagliarone
Everton dos Santos
Giuliano Bonfá
Non-living antigens vaccines
(purified or recombinant subunits)
Advantage : safety (no possibility of disease development)
Adjuvants enhance the strength and duration of
Disadvantage:
immunogenicity
immunepoor
responses
and modulates the type of
immune response to the vaccine antigen
ADJUVANTS !!!!
Adjuvant : additional vaccines component that enhances the
immune response to antigens in vivo .
Vaccine antigen
Adjuvant
amplification of immune cells signals
Immune response
A
B
Signal 0 : antigen recognition by DC leads to
cell activation and maturation
Signal 1 : DC presents antigen peptides through MHC class II molecule
to the TCR of naive CD4+T cell
Signal 2: DC expresses high levels of MHC, co-stimulatory (CD40, CD80
and CD86) and adhesion molecules (CD54 and CD58).
C
CD4+ T cell
differentiation
Signal 3 : DC secretes high amounts of cytokines that are
crucial for inflammation and differentiation of
CD4+ T cells.
(modified)
ADJUVANTS
DELIVERY SYSTEMS
-“Antigen vehicles”
-Carriers to which antigens are associated
by way of adsorption,co-precipitation or
encapsulation.
-Immunogenicity :
- antigen retention at site of injection
(antigen depot effect);
- increase of uptake by DC cells and
macrophages (slow release of antigen)
IMMUNOMODULATORS
-Tipically small molecules with adjuvant
funtions through other mechanisms
than antigen retention;
-Immununogenecity:
Direct stimulation of innate immune cells
through interaction with PRRs TLRs,NLRs,
RLRs and C-type lectins.
*
* More used in human vaccines
Adjuvant action of aluminium salts
Adjuvant mechanism of aluminium salts : antigen precipitaded onto
insoluble particles of these salts are released slowly in the body
(“depot effect”-Alexander Glenny, 1926).
HOWEVER .....
Studies have shown that the adjuvant activity of aluminium is
more complex than this.....
Human macrophages pre-immunized with toxoid tetanus + aluminium
hydroxide and further co-cultured with autologous T cells
Increased IL-1 production and T cell proliferation
Aluminium can activate antigen-specific
immune response by activating APCs!
How aluminium salts can stimulate APCs and adaptive
immune responses ?
- In culture of lymph node cells from mice immunized with alum it was observed
increased IL-1 and IL-4 production and proliferation of T cells. Treatment with
anti-IL-4 decreased the proliferation (GRUN & MAURER, 1989)
-Immunization with alum in mice enhaced only Th2 antibodies (IgG1 and IgE).
However, IL-4-/- mice presented induced IgG2a and Th1 cytokines production
(BREWER et al.,1996)
Aluminium hydroxide induced chemokines secretion ( CCL2/3 and 4 and
CXCL-8) and inhibited CD14 expression in human monocytes and enhances
MHC II and CD86 expression in DCs (monocyte activation, recruitment
to blood and differentiation toward DCs.) (SEUBERT et al., 2008).
DCs activation by aluminium salts occurs through
specific receptors???????
MyD88-deficient mice immunized with antigen and aluminum salts produced
IgG1 and specially more IgE, comparing to control mice.
Deficient micefor both MyD88 and TRIF immmunized with alum + TNP-Hy showed
Th2-induced IgG1 and IgE responses comparable to those observed in control mice.
According to these observations, aluminium salts are both
delivery systems and immunomodulators adjuvants,
although their activities do not occur through TLRs....
IMMUNOMODULATORS
- Are tipically small molecules that exert their adjuvant funtions through
mechanisms other than antigen retention
Direct stimulation of innate immune cells (monocytes, macrophages, NK and
NKT cells and DCs) specially through interaction with PRRs of these cells (TLRs,
NLRs, RLRs and C-type lectins
PAMPs
However, its known that there are ENDOGENOUS compounds which are also
able to be recognized by host cells, inducing the immune response even in
absence of pathogens.
DAMPs
Damage
Associated
Molecular
Patterns
DAMPs = substances/compounds released by necrotic cells which are recognized by
innate immune cells (tissue injury indicators). They activate innate immune cells
according to the Danger Hypothesis.
Are DAMPs endogeous adjuvants ?
Dead cells co-administered with antigen induced increased
antigen-specific CD4+ T cells in vivo. Moreover, dead cells
could lead to DCs maturation in vitro.
Uric acid stimulates DC maturation in vitro and
stimulates CD8+ T cell response in antigen- immunized
mice
How DAMPs are recognized by DCs ?
Recognition of endogenous DAMPs by TLRs
There are studies that defend the idea of TLR ligands contamination (such as LPS) in studied
DAMPs, others showed that TLR knockout mice presented reduction in inflammatory
response to necrotic cell death in vivo (CHEN & NUÑEZ, 2010).
MSU: monosodium urate
Human monocytes
MRU-induced
peritonitis in mice
Human monocytes
Mechanisms for DAMPs –induced inflammation
cell recruitment
DAMPs and PAMPs are recognized by innate immune cells leading to further
adaptive cells activation (adjuvant activity!!).
Aluminium adjuvants can indirectly activate APCs by causing
tissue damage due to necrosis of skeletal muscle fibers (SHY et al., 2003)
Uric acid is able to induce NLRP3 (MARTINON et al., 2006)
Aluminium adjuvants can activate the inflammasome NALP 3?
Mice submited to OVA or OVA+alum
injections i.p.
Nalp 3or ASC 3 or Casp 3 deficient mice injected i.p with OVA
Possible mechanisms of alum action
Indirect activation
ex: TLRs
Direct activation
Everton dos Santos
Giuliano Bonfá
Show that silica crystal and aluminum induces
activation of MAPK beyond of NALP3
Everton dos Santos
Could silica and alum induce inflamossome activation of macrophages?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
ELISA (IL-1β)
(culture Supernatants)
Macrophages - Silica ( 50 -100µg/mL) ;
(5x105/mL)
- Aluminium (200-400µg/mL);
- ATP (1mM).
Silica, alum and ATP induced inflamossome activity on LPS-primed macrophages
Besides of IL-1B production, could macrophages produce others inflammatory
factors?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
Macrophages - Silica ( 50 -100µg/mL) ;
(5x105/mL)
- Aluminium (200-400µg/mL);
ELISA (PGE2, TNF-α, IL-1β, 6, 12,
18)
(culture Supernatants)
- ATP (1mM).
These results showed that in addition to IL-1β and IL-18, macrophages also produce PGE2
in response to silica, alum and ATP.
Can DCs produce IL-1β and PGE2 in response stimulation for silica or alum?
GM-CSF (10ng/mL)
DCs
(5x105/mL)
Stimulation for 6h
ELISA (PGE2,IL-1β)
- Silica (100µg/mL) ;
(culture Supernatants)
(1ng/mL)/3h
BM cells
M-CSF (10ng/mL)
Primed with LPS
DC cells
(5x105/mL)
- Aluminium (400µg/mL);
Macrophages
(5x105/mL)
Macrophages
(5x105/mL)
DCs can produce IL-1β and PGE2, but macrophages produce higher amounts of PGE2
than DCs
*B6/Balb; PBMC
In short...
Aluminium, Silica
Macrophages
MB;
Peritoneal
DC
MB;
 Activity inflammasome;
 Production of IL-β
 Prostaglandin E2
Does is necessary the phagocytosis for production of IL-1β and prostaglandin?
Could the engulfment of particulates, lysosomal rupture, and release of
lysosomal enzymes induce PGE2 and IL-1β production in macrophages?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
ELISA (PGE2, IL-1β)
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
-
Alum (400µg/mL);
-
Cyt D (2μM or Cat B (10μM)
(culture Supernatants)
Phagocytosis, lysosomal damage and release
of enzymes triggers PGE2 production in
macrophages.
In short...
Phagocytosis
Aluminium, Silica
Macrophages
lysosomal rupture
release of lysosomal enzymes
prostaglandin E2 and IL-1β production
Does prostaglandin production is dependent of inflammasome?
The production of PGE2 is dependent of activity of inflammasome?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
- TiO2 (100µg/mL);
ELISA (PGE2, IL-1β)
(culture Supernatants)
The production of PGE2 is dependent of activity of inflammasome?
Primed with LPS
M-CSF (10ng/mL)
WT; Nalp3, Asc,
Casp1 (-/-)
(1ng/mL)/3h
BM cells
ELISA (PGE2)
Stimulation for 6h
Macrophages
(5x105/mL)
-
Silica (50μg/mL)
-
-Alum (200μg/mL)
(culture Supernatants)
Aluminium, Silica
Macrophages
MB;
Peritoneal
PGE2 production in macrophages is independent of
inflammasome

Activity inflammasome;

Production of IL-β

Prostaglandin E2
In short...
Phagocytosis
Aluminium, Silica
Macrophages
lysosomal rupture
release of lysosomal enzymes
prostaglandin E2 and IL-1β production
The PGE2 production was independent of
inflammasome activity.
Prostaglandin E2 (PGE2) is generated by the sequential metabolism of arachidonic acid by
cyclo-oxygenase and prostaglandin E synthase (Needleman et al., 1986; Smith. 1992).
Could alum and silica induce the activity of cyclooxygenase (COX) and
synthase PGE?
Could silica and alum induce activity of COX or PGE synthase enzymes?
Primed with LPS
WT
PTGES (+/+;
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
ELISA (PGE2, IL-1β)
(culture Supernatants)
- COX-2 inhibitor (1µM);
-/-)
Silica- and alum-induce PGE2 production in macrophages mediated by the COX-2 and
PTGES.
In short...
Phagocytosis
Aluminium, Silica
Macrophages
lysosomal rupture
release of lysosomal enzymes
COX-2 and PTGES
Activation of the inflammasome
Prostaglandin E2 production
IL-1β production
Which signaling pathway is involved in this process?
Which signaling pathway is involved in the production of PGE2?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
ELISA (PGE2, IL-1β)
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
-
(culture Supernatants)
Absence of inhibitors were used as
the 100% controls
Inhibitors
These results showed that P38 MAP
kinase is involved with in the PGE2
production
and
rupture
necessary
is
activation.
the
lysosomal
for
your
In short...
Phagocytosis
Aluminium, Silica
lysosomal rupture
Macrophages
release of lysosomal enzymes
Phospholipase A2???
P38 MAPK phosphorilation
COX-2 and PTGES
Prostaglandin E2 production
Activation of the inflammasome
IL-1β production
Phosphorylation of p38 MAPK could activate phospholipase A2 after
phagocytosis of silica or aluminium by macrophages?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
-
Inhibitors
ELISA (PGE2, IL-1β)
(culture Supernatants)
Absence of inhibitors were used as
the 100% controls
Phosphorylation of p38 MAPK active
phospholipase A2
The Syk can be activated by phagocytosis
de silica or aluminum??
The Syk can be activated by phagocytosis de silica or aluminum?
Primed with LPS
Macrophages
(5x105/mL)
(1ng/mL)/3h
Stimulation for 2h
- Silica (100µg/mL) ;
Macrophages
(5x105/mL)
-
ELISA (PGE2, IL-1β)
or blot
Inhibitors
These results suggest that lysosomal damage triggers Syk activation, and then activated
Syk upregulates cPLA2 activity via the phosphorylation of p38 MAP kinase.
Phagocytosis
In short...
lysosomal rupture
Aluminium, Silica
Macrophages
release of lysosomal enzymes
Syk
P38 MAPK phosphorilation
Phospholipase A2
COX-2 and PTGES
Prostaglandin E2 production
Activation of the inflammasome
IL-1β production
- PGE2 inhibits production of cytokines such as TNF-α,
and IL-12 (Scales et al., 1989; Van der Pouw Kraan et al.,
1995).
- PGE2 may polarize cellular response toward a Th2
phenotype enhancing IL-4 and IL-5 production (Betz
Prostaglandin
synthases
and Fox, 1991; Katamura et al., 1995) and facilitating
immunoglobulin class switching to IgE (Roper et al.,
1995).
- PGE2 modulates the functions of cell populations, such
as T cells and macrophages (Nataraj et al., 2001).
Silica- and Alum-Induced Production of PGE2 by Macrophages Regulates
Immune Responses In Vivo?
Silica- and Alum-Induced Production of PGE2 by Macrophages Regulates
Immune Responses In Vivo?
OVA+Alum or OVA+Silica
0
7
17
Sera collected
Days
Ptges
+/+
or Ptges
ELISA
IgG1, IgG2c, IgE
-/-
PGE2 production but not inflammasome activation in macrophages, positively
regulates the generation of IgE antibodies in vivo.
Phagocytosis
Conclusion
lysosomal rupture
Aluminium, Silica
Macrophages
release of lysosomal enzymes
Syk
P38 MAPK phosphorilation
Activation of the inflammasome
IL-1β production
Phospholipase A2
A.A
COX-2  PTGES
Prostaglandin E2 production
IgE
Aim: To identify the citotoxic and adjuvant effect of alum on immune
responses
Is alum cytotoxic on local of injection?
i.m. and i.p.
OVA + Alum
days 0, 14 and 21
Peritoneal lavage fluid
Muscle lavage fluid
Cell death rate
i.p.
Alum induce cell death and release of host DNA at sites of
injection!
Can DNA act as an adjuvant?
i.p.
OVA
OVA + Alum
OVA + DNA
i.p.
ELISA
days 0, 14 and 21
OVA
OVA + Alum
days 0 and 10
DNase I
(3 and 8h)
ELISA
Can DNA act as an adjuvant?
OVA-specific CD4+
CFSE
i.p.
OT-II
OVA
OVA + Alum
OVA + DNA
DNase I
(3 and 8h)
3d
Cells on the bronchial
lymph nodes (BLNs)
WT
Host DNA released by alum cytotoxicity mediates alum
activity on humoral and TH2 cell responses!
What’s the mechanism?
• TLR9-/- mice develop humoral responses similar to those of their WT
counterpart in response to alum immunization;
NALP3-/- and
CASP1-/-?
Nature Reviews Immunology 10, 123-130 (February 2010) | doi:10.1038/nri2690
What’s the mechanism?
i.p.
OVA
OVA + Alum
OVA + DNA
days 0, 14 and 21
i.p.
ELISA
Peritoneal lavage
fluid
NALP3
independent
OVA
OVA + Alum
OVA + DNA
ELISA (serum)
days 0, 14 and 21
WT and IRF3-/-
What’s the mechanism?
i.p.
OVA
OVA + Alum
ELISA (serum)
days 0, 14 and 21
TBK1+/-/TNF-/and
TBK1-/-/TNF-/-
Alum and host genomic DNA trigger type I IFN secretion and
IgE responses through activation of the TBK1-IRF3 axis!
Have IRF3 deficiency an effect on Th2
responses after alum imunization?
OVA-specific CD4+
CFSE
i.p.
OT-II
OVA
OVA + Alum
OVA + DNA
3d
Cells on the bronchial
lymph nodes (BLNs)
WT and IRF3-/-
IRF3 is essential for the boosting of canonical TH2 cells by
alum and genomic DNA!
There is an effect during experimental asthma?
i.p.
OVA
OVA + Alum
days 0 and 14
WT and
Bronchoalveolar lavage
fluid (BALF)
IRF3-/Airway
sensitization
d 21 to 25
IRF3 absence protect mice from allergic airway
inflammation!
IRF3 deficiency changes DCs migration?
i.p.
OVA
OVA + Alum
OVA + DNA
24 - 48 h
WT and IRF3-/-
FACs (peritoneal lavage fluid and BLNs)
• iMonos (F4/80int CD11b+ Ly6C+ Ly6G- cells)
• cDCs (MHCII+ CD11c+ F4/80low Ly6C- cells)
• pDCs (B220+ Ly6G+ CD11cint F4/80low cells)
BLNs
The recruitment of iDCs to the BLNs of alum-treated mice strongly correlated with the percentage of cell death and
DNA release and that it was reduced after Dnase I treatment.
IRF3 is essential for the triggering of iDC recruitment by
alum!
Deficiency on DCs recruitment alter the Th2
response?
OVA-specific CD4+
i.p.
CFSE
OT-II
OVA
OVA + Alum
Cells on the bronchial
lymph nodes (BLNs)
WT and IRF3-/WT immunized iMonos
IgE  increase
IgG1  not affected
Deficient migration of inflammatory monocites (iMonos)
impair alum-induced Th2 and IgE responses in IRF3-/- mice!
IRF3 deficiency changes iDCs activation?
i.p.
OVA
OVA + Alum
FACs and ELISA (peritoneal
lavage fluid and serum)
days 0, 14 and 21
WT and
IRF3-/-
iMonos (F4/80int CD11b+ Ly6C+ Ly6G- cells)
BLNs
IgE  attenuated
IgG1  not affected
Alum-induced iMono migration depends on IL-12p40
homodimer signaling!
Conclusion
DAMP
DAMP
Journal Conclusion
Alum
NALP3
Independent
NALP3
Dependent
PGE2
Dependent
Host DNA (DAMP)
Uric acid crystals
(IRF3-TBK1 axis)
Syk and p38 MAP kinase
Th2 responses – IgE and IgG1