IMMUNOLOGIC ADJUVANTS-2
CONTENTS
•
•
•
•
Licensed Adjuvants
Functions
Immunological Perspectives
Alums and Aluminium Salts
ADJUVANTS
An adjuvant is a substance that, when added to a
vaccine, greatly enhances its protection against infection.
Ajuvare - to help (Lt.)
Help in:
• Decreasing the amount of antigen required for activation
• Safe
• Effective
• Lower number and amount of vaccine doses
WHY USE ADJUVANTS?
Adjuvants have been traditionally used to increase the magnitude of an adaptive
response to a vaccine. The targets, therefore, are to:
(1)increase the response to a vaccine in the general population, increasing mean
antibody titers and/or the fraction of subjects that become protectively immunized;
(2)increase seroconversion rates in populations with reduced responsiveness
because of age (both infants and the elderly), disease, or therapeutic interventions, as
in the use on the MF59 adjuvant to enhance the response of older subjects to
influenza vaccine;
(3)facilitate the use of smaller doses of antigen because the ability of an adjuvant
to permit comparable responses with substantially lower amounts of antigen could be
important in circumstances in which large-scale vaccination is urgent and production
facilities limiting, as in the emergence of a pandemic influenza strain; and
(4)permit immunization with fewer doses of vaccine
WHY USE ADJUVANTS?
The adjuvants now can even direct the adaptive response towards a particular
immune arm. This is done to:
(1)provide functionally appropriate types of immune response (e.g., T
helper 1 [Th1] cell versus Th2 cell, CD8+ versus CD4+ T cells, specific
antibody isotypes);
(2)increase the generation of memory —especially T cell memory
(3)increase speed of initial response, which may be critical in a pandemic
outbreak of infection; and
(4)alter the breadth, specificity, or affinity of the response
EFFCTIVENESS OF ADJUVANTS
IMMUNOLOGY OF ADJUVANTS
Adjuvants, in general, present their effects by adapting/ manipulating 4 main
mechanisms:
1.
2.
3.
4.
Depot effect
PRR Activation
Inflammosome activation
MHC presentation
LICENSED ADJUVANTS
ALUMINUM ADJUVANTS
•Aluminum Hydroxide
•Aluminum Phosphate and
•Alum
ALUMINIUM HYDROXIDE
STRUCTURE AND PROPERTIES
Aluminum hydroxide is not Al(OH)3 but rather crystalline aluminum
oxyhyroxide (ALOOH)
This difference is important because crystalline aluminum hydroxide has low
surface area i.e., ~20-50 m2/g
Also is a poor adsorbent
• Crystalline aluminum oxyhydroxide has a surface area of
~500m2/g and thus this makes it a good adsorbent
• This high surface area is due to its morphology
• Primary particles are fibers having dimensions of 5 x 2 x
200nm
• The crystalline nature also allows identification based on x-ray
diffraction
• The surface is composed of Al-OH and Al-O-Al groups
• Where the Al-OH group can either act as a proton acceptor or
donor
• Iso electric point of Al-OH is 11.4
• Thus the adjuvant exhibits a positive surface charge at pH 7.4,
the pH of the intestinal fluid
• Aluminum hydroxide is soluble in acidic media below pH 4
• Also in basic media pH above 10
• It is also soluble in neutral pH solutions of a-hydroxy acids
such as citric acid
ALUMINUM PHOSPHATE
• Aluminum phosphate is chemically amorphous aluminum
hydroxyphosphate in which of the hydroxyl groups are
replaced by phosphates
• The primary particle has a plate like morphology with a
diameter of ~ 50nm
• The surface is composed of Al-OH and Al-OPO3 groups
• Iso electric point varies from 9.4 to 4.5 depending upon the
degree of phosphate substitution
ALUM
• Water soluble and chemically is aluminum potassium
sulphate AlK(SO4)2
• In earlier vaccines a solution of alum was mixed with
a solution of the antigen dissolved in a phosphate
buffer
• It is common practice to refer to the adjuvant
produced by in situ precipitation as ALUM
CONTENT UNIFORMITY
• The morphology of aluminum hydroxide and aluminum
phosphate adjuvant provides a mechanism that uniformly
distributes microgram quantities of antigen onto milligram
quantities of adjuvant
• The functioning unit of both adjuvants is an aggregate ranging
in size from 2 to 20 um composed of the nanometer sized
primary particles
EFFECT OF FREEZING
• Vaccines containing the aluminum salt adjuvants should not be
allowed to freeze
• Freezing can affect both the adjuvant and the antigen
• Freezing causes coagulate formation in the formulations that
cannot be redispersed
• Freezing can also cause changes in the secondary, tertiary or
quaternary structure of the antigen
CLEARANCE OF ALUMINUM SALT
ADJUVANTS
Aluminum adjuvant containing vaccines are usually
administered by intramuscular or subcutaneous
injection
Thus if they come in contact with the intestinal fluid the
activity is altered.
Intestinal fluid contains 3 hydrocarboxcylic acids
1. Citric acid
2. Malic acid and
3. Lactic acid
These are good chelators of aluminum and thus solubilize
the adjuvants
ALUMINIUM ABSORPTION IN BODY
The absorbance and clearance of aluminum adjuvants was studied in
rabbits with labeled aluminum 26 (isotopic form)
Thus any Al26 in the blood, urine or tissue following intramuscular
administration is from the dissolution of the adjuvant in intestinal
fluid
This can be then accurately quantified by mass spectrometry
Al26 was found in the blood after 1 hr of adjuvant administration,
depicting that dissolution of the adjuvant begins upon
administration, it was also shown that 51% of adjuvant had
dissolved in the intestinal fluid and had been absorbed in the blood
in 28 days
ADSORPTION MECHANISM
• Major mechanisms for adsorption are
• Electrostatic attraction
• Hydrophobic forces and
• Ligand exchange
• Electrostatic attraction being the most frequently
utilized mechanism
• This attraction can be optimized by determining the
iep of the antigen and then selecting an adjuvant that
will have the opposite surface charge at the desired
pH.
• For example: at pH 7.4 aluminum hydroxide adjuvant
iep 11.4 adsorbs albumin iep 4.8 but does not adsorb
lysozyme iep 11.0
• In contrast aluminum phosphate iep 4.0 adsorbs
lysozyme but not albumin
Care must be taken in selecting a buffer for an aluminum
hydroxide adjuvant containing vaccine
Acetate and Tris are buffers that do not alter the iep of
aluminum hydroxide adjuvant
The ionic strength of the vaccine is very important when
adsorption is due to electrostatic attractions
The ionic strength of the vaccine should be kept as low as
possible if adsorption is through electrostatic attraction
Hydrophobic forces can also contribute to the adsorption of antigen
by aluminum adjuvants
The contribution of these forces can be determined by observing the
effects of ethylene glycol on adsorption. The adsorption of lysozyme
by aluminum phosphate adjuvant is reduced by the addition of
ethylene glycol
Ligand exchange a relatively lesser used mechanism helps in the
binding by exchange of functional groups
DESORPTION IN INTESTINAL FLUID
• Vaccines come in contact with the intestinal fluid
• The composition of the intestinal fluid is very different
from the vaccine
• Invitro studies have identified several components of
the intestinal fluid all of which can cause rapid elusion
of the antigens. These are
• Phosphate
• Citrate
• Fibrinogen, etc
• Lysozyme was shown to be completely eluted from the
aluminum phosphate adjuvant after 15 minutes in
sheep intestinal fluid
MECHANISM OF ACTION OF
ALUMINUM SALT ADJUVANT
It is surprising that there is no consensus regarding the mechanism
by which aluminum containing adjuvants potentiate the
immune response
Three potential mechanisms are frequently cited to explain how
these salt adjuvants increase antibody production
1.
Depot mechanism
2.
Inflammation mechanism
3.
Promotion of uptake of antigen by APCs
DEPOT MECHANISM
This mechanism postulates that the aluminum containing adjuvant
and the adsorbed antigen remain at the site of injection
The antigen is released slowly to stimulate the production of
antibodies
INFLAMMATION MECHANISM
This mechanism is based on the hypothesis that aluminum
containing adjuvants cause inflammation at the site of injection
APCs are rapidly attracted to this site
And since the antigen is also present at the site of injection APCs
encounter a high concentration of antigen
PROMOTION OF ANTIGEN UPTAKE BY APCS
• It has also been proposed that adsorption of antigen to
aluminum containing adjuvants converts the soluble
antigen to particulate form
• APCs can uptake the particulate matter by
phagocytosis
• Thus the antigen remains adsorbed, is taken into the
macrophages and DCs and then further processed
• It is likely that all 3 proposed mechanisms contribute to
the initiation of immune responses
THANK YOU