Immunologic Adjuvants

INTRODUCTION• Added into vaccines to increase the

immunogenicity of the antigen• Immunogenicity ???• 1925 Ramon (scientist) Diphtheria antitoxin

in horses• Discovered that adjuvants can non specifically

enhance the immune response• The term “ADJUVANT” coined by Ramon comes

from a Latin word adjuvare meaning to help or aid.• 1926 Glenny and colleagues Aluminum salts• Commonly termed as ALUM Adjuvants• The only adjuvant used in liscenced vaccines in

the US• And the most commonly used adjuvant in the rest

of the world.

Classification of Immunologic Adjuvants

• Mineral salt Aluminum hydroxide • Microbial derived Bacterial toxins

DNAFlagellin

• Particulate Biodegradable polymerLiposomesVirosomes

• Oil emulsion and surfactant basedFreund’s adjuvant• Synthetic Polynucleotides

LPS based adjuvants• Cytokines IL2, IL12, GM-CSF

IFN gamma• Genetic Cytokine genes

genes coding co- stimulatory

molecules

Adjuvant Mechanism of ActionAdjuvants in Acquired and Innate Immunity

Immune system

Innate Acquired

Innate immune system• First line of defense• Acts rapidly• Activates pathogen specific defenses• Through phagocytic cells such as macrophagesAcquired immune system• Mounted over days to weeks post infection• But generates memory cells helpful for future

encounters

• Previously thought that innate immune system acts non specifically and independent of acquired immune system

• Now established that innate immune system responds specifically to types and classes of pathogens

• Also, it guides and initiates appropriate acquired immune responses

Primary mechanism• Ability of the innate immune system to

recognize pathogen specific molecular patterns (PAMPs) through pattern recognition receptors (PRRs)

• Innate system recognizes PAMPs as “Danger Signals”

• Triggering the initiation of acquired responses• Primary mechanism of microbial based

immunologic adjuvants is to present PAMPs in association with protective antigens ultimately resulting in induction of acquired immune system

• Therefore PAMPs based vaccines are especially suitable for highly purified vaccines and subunit vaccines that may lack sufficient recognition by the host

• The most well studied PRRs used by immunologic adjuvants are the Toll like receptors.

Induction of Immunomodulatory Chemokines and Cytokines by

Adjuvants

• The result of binding of the adjuvant to the PRRs of the host is the induction of proinflammatory cytokines and chemokines

• Chemokines attract phagocytic cells to the site of infection or in terms of vaccines at the site of injection.

• These can then act directly or indirectly on helper lymphocyte subsets to modulate acquired immune responses

• TH1• TH2

Effects of Adjuvants on Antigen Presenting Cells

• Dentritic cells are the principal antigen presenting cells for priming immune responses to vaccine antigens and are the only APC that can present antigen to naïve T cells

• Dentritic cells are a primary cellular link between the innate and the acquired immune system

• Adjuvants can enhance antigen uptake processing and presentaion

by various types of dentritic cells• Evidences suggest and confirm that adjuvants

are internalized after intramuscular injection by dentritic cells in mice.

Effects of Adjuvants on Antigen Delivery

• Adjuvants that are not presently known to function by mechanisms directly involving PAMP recognition are alums, calcium salts and emulsion based adjuvants

• They act principally through antigen delivery and presentation mechanisms and have been classified as antigen delivery systems

• Mechanism of action is the so called “Depot effect”• Mineral salt adjuvants and emulsion based

adjuvants associate with the antigen and effectively increase its biological and immunologic “half life” at the site of injection

• Also, they improve delivery of antigen to APCs and to the secondary lymphoid organs.

• The immunogenicity of synthetic peptides and the soluble antigens that are otherwise cleared by the body without sufficient delivery to the lymph nodes can be improved using these adjuvant.

Types of Immunologic Adjuvants

• Aluminum Adjuvant• CpG Oligodeoxynucleotides• Cytokine Adjuvants• MF59 Adjuvant• Monophosphoryl Lipid A (MPL)

Adjuvant• Mucosal Adjuvants

CpG Oligodeoxynucleotides • Bacterial DNA is recognized as PAMP in

mammalian cells• Serves as a potent activator in the innate

immune system through interactions with TLR9

• Fragments of bacterial DNA and synthetic single stranded oligonucleotides containing unmethylated CpG motifs (CpG ODN) found in bacterial DNA have also been shown to be powerful adjuvants.

• In humans CpG motifs are recognized by TLR9 found on NK cells, B cells and DCs.

• Stimulation by CpG also leads to maturation and activation of APCs

• The use of CpG ODN have been shown to act as adjuvants for antigens delivered through the mucosal or parentaral route

• Preferrentially induce TH1 over TH2 responses

• Also, “redirect” immune responses that have a natural TH2 bias

• Experiment by Weeratna et al• HBV surface antigen coupled with either

alum or CpG ODN adjuvant

• CpG resulted in TH1 bias• Alum resulted in TH2 bias• Mice primed with alum and later

boosted with CpG resulted in TH1 bias

• Mice primed with CpG ODN and later boosted with alum maintained initial TH1 bias

• These results suggest that CpG ODN could redirect a TH2 response towards a TH1 bias in the HBsAg model

Cytokine Adjuvants• IFN gamma• IL2• INF alpha• IL12• Adjuvant activity of IL12 has been

demonstrated via Leishmania vaccine in mice

• Immunization of Balb/c mice with L. major antigens and IL12, induced leishmania specific CD4+ TH1 cells and conferred protection against L. major.

• Immunization of control animals with antigen alone elicited a TH2 response that were not protective

• Cytokine mixtures including GM-CSF, INF alpha and IL12 emulsified with incomplete Freund’s adjuvant have also been employed to “steer” the immune response towards a TH1 bias vs a TH2 bias

• In these studies it was demonstrated that in Balb/c mice GM-CSF acted in synergy with IL12 to produce cytotoxic T cell response and the suppression of TH2 cytokines IL4 and IL5 when delivered with HIV 1 peptide antigens in incomplete Freund’s adjuvant

MF59 Adjuvants• Oil in water microfluidized emulsion

composed of• Stable droplets (250nm) of the metabolizable

oil squalene• Two surfactants• Tween 80 and • Span 85• Novartis vaccines developed MF59 to be an

adjuvant for human vaccines with higher potency than alum and equally low toxicity

• MF59 is used in FLUAD an influenza vaccine licensed in Europe

• Efficacy of the vaccine with and without the adjuvant was demonstrated in mice

• It was seen that MF59 significantly increased the antibody response to influenza antigens over a wide dose range

• In terms of safety and tolerability the adjuvant vaccine produced more local reactions than the conventional one but these were mild and subsided after 2-3 days of immunization

• MF59 is a safe, practical and potent adjuvant for use with human vaccines

• The formulation is easily manufactured, may be sterilized by filtration and is both compatible and efficacious with all antigens tested to date

• It has been shown to be a potent activator of both cellular and antibody immune responses

• Currently in use with HSV, HIV, influenza and many other vaccines

Monophosphoryl Lipid A (MPL) Adjuvant

• Johnson and colleagues discovered the adjuvant effects of lipopolysaccharide endotoxin from gram negative bacteria in 1956

• Since then efforts are going on for the identification of native LPS like adjuvants or its adjuvant active moiety Lipid A, one that would be devoid of its toxicity

• MPL adjuvant (GSK Bio) is a monophosphoryl lipid A prepared from the lipopolysaccharide of a mutant of Salmonella minnesota

• Retains the immunomodulatory effect coupled with reduced toxicity

• Because of its safety and extensive clinical evaluation, MPL is a strong candidate for use as an adjuvant for human preventive and therapeutic vaccines against infectious and neoplastic diseases

Vaccine antigens tested with MPL include

• Recombinant proteins• Polysaccharide conjugates• Allergens and• Tumor antigens

Virosomes

• Virosomes are spherical, vesicles with mean diameter of 150 nm containing 70% egg yolk phosphatidylcholine and 20% synthetic phosphoethanolamine

• The influenza virus hemagglutinin (HA) and trace quantities of neuramidase (NA) are intercalated within the phospholipid bilayer

Virosome Components

• The role of the HA in the preparation of these vesicles appears to be that of binding to sialic residues on the cells to enhance endocytosis and fusion of the virosome with endosomes

• The objective is to enhance the uptake of the associated vaccine and its resulting immunogeneicity

• Several vaccines have been formulated with virosomes and tested for their safety and immunogenicity in clinical trials in humans these include:– Hepatitis A virus vaccine– Influenza subunit vaccine– Diptheria and tetanus toxoids

• Virosome-adjuvanted vaccines against hepatitis A virus and influenza have been licensed in several countries

Mucosal Adjuvants

• Formulation of vaccine antigens with several types of immunologic adjuvants active at the mucosal surfaces have been shown to facilitate the delivery of vaccines via the oral or nasal route of administration rather than the traditional precutaneous injection

Examples• Cholera toxin B subunit• Cholera holotoxin• Mycoplasma derived macrophage activating lipopeptide, etc

Mutants of Bacterial Toxins as Mucosal Adjuvants

– E. coli heat labile enterotoxin (LT)– Cholera toxin (CT)

• These toxin consist of two subunits – A subunit: contains enzymatic activity– B subunit: binds to other glycolipids

• Their use in humans is limited by their ability to cause diarrhea in recipients

• The most successful approach to overcome this problem is to develop nontoxic derivatives with site-directed mutagenesis

• Several mutants have been produced which behave as strong mucosal adjuvants in mice and other animal models when administered with:– recombinant proteins – synthetic peptides– other vaccine constructs

Oil Emulsions• Freund's adjuvant consists of a water-in-oil

emulsion of aqueous antigen in paraffin (mineral) oil of low specific gravity and low viscosity

There are two Freund's adjuvants: – The incomplete Freund's adjuvant

consists of water-in-oil emulsion without Mycobacteria

– The complete Freund's adjuvant consists of the same components but with 5 mg of dried, heat-killed Mycobacterium tuberculosis added