IMMUNOLOGIC ADJUVANTS-2

Licensed Adjuvants Functions Immunological Perspectives Alums and Aluminium Salts CONTENTS

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 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 WHY USE ADJUVANTS?

EFFCTIVENESS OF ADJUVANTS

Adjuvants, in general, present their effects by adapting/ manipulating 4 main mechanisms: 1.Depot effect 2.PRR Activation 3.Inflammosome activation 4.MHC presentation IMMUNOLOGY OF ADJUVANTS

LICENSED 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 ALUMINUM ADJUVANTS

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

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 CONTENT UNIFORMITY

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 EFFECT OF FREEZING

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 CLEARANCE OF ALUMINUM SALT 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

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 ADSORPTION MECHANISM

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

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 DESORPTION IN INTESTINAL FLUID

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 MECHANISM OF ACTION OF ALUMINUM SALT ADJUVANT

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 DEPOT 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 INFLAMMATION MECHANISM

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 PROMOTION OF ANTIGEN UPTAKE BY APCS