Recent advances in Vaccine Adjuvants

Dr P. Swain, Senior scientist,

Fish Health Management DivisionCentral Institute of Freshwater Aquaculture,

Bhubaneswar.

A vaccine is a biological preparation that produces an adaptive immunity to a particular disease/condition and typically contains antigen/protein that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins or a form of protein .

What is a “Vaccine”

How Vaccines Work

1. The general answer

Vaccines work by fooling the body into thinking it is infected with a antigen so that next time when it sees the real antigen it will be ready faster with a more powerful responseSometimes it gets the body to do something different and better then if it were naturally infected

We recognize them as something different not belonging inside the bodyOnce recognized we try and kill themWe have two systems of doing this:

The Innate immune systemThe adaptive immune system

2. The Battle Between Us and pathogens: What we can do

How Vaccines Work

ThymusMucosal associated lymphoid tissues(MALT)IgM antibodyLittle evidence of immunological memoryLack of bone marrow and lymph nodeMelano macrophage centres

ThymusHaematopoitic tissues in bone marrowIgM amd IgGEvidence immunological memory

Amphibia

Fish

Reptiles

Thymus

Haematopoitic tissues in bone marrow

MALT and lymph nodes

IgM amd IgG like antibody

Evidence immunological memmory

Also have Bursa-like clocal lymphoid tissues

BirdsThymusWell defined Bursa fabricius- developes earlyRapid memmory responseIgM, IgG and IgA antibodyAllergic response involving an IgE sub class

Mammals

ThymusBone marrow for B cellGreater diversity in immunoglobulinMemoryAllergic response involving an IgE sub class

Immunoglobulin evolution

Types of vaccine

1. Live, Attenuated (weakened)

Vaccines

A. First generation

vaccines

2.

Inacti

vated

(killed)

Vaccines

B. Second generation vaccines

3.Conjugate Vaccines (is created

by covalently attaching a poor antigen to carrier

protein.)

4. Subunit Vaccines

(Antigens of microbes to

stimulate response)

5. Recombinant Vaccines (a section of DNA from one species is inserted into the DNA of another).

C. Third generation vaccines

DNA vaccines

Peptide(synthetic)vaccines

WORLD HEALTH ORGANISATION: A REPORT

"Nearly nine million children under 14 years of age die every year from infectious disease. And at

least a third of them could be saved if existing vaccines were more widely used, but the rest only if

suitable new vaccines were developed..."

Good immune response Both Cell Mediated Immunity and antibody responses. Immunity is long lived Single dose Safety Danger of reversion to virulence, or Severe disease in

immunocomprised Stability Organisms in the vaccine must remain viable in order to infect and

replicate in the host Vaccine preparations are therefore very sensitive to adverse storage

conditions Maintenance of the cold chain is very important. Expense Cheap to prepare

Inefficiency of currently used vaccines

New generation vaccines are vaccines mainly contain purified proteins, peptides, recombinant proteins which are poorly immunogenic. Inability to elicit cell-mediated immune responses. poor self life and highly unstableDegradation by the host enzymatic systems

Therefore, the search for harmless and effective adjuvants is in process

Why Adjuvants?

In many cases, the antigen itself is only very weakly immunogenic; therefore, an adjuvant is needed to intensify the immune response

Adjuvants can also be included in vaccines to guide the type of immune response generated (cancer, HIV or mucosal immunizations)

The adjuvant may facilitate targeting and/or controlled release of the antigen to antigen presenting cells

Adjuvants and delivery vehicles have also been shown to protect antigens from degradation

Adjuvants are…….

Adjuvants are components that are added to vaccine antigen to make them more immunogenic.

How do adjuvants work?

Antigen depot formation Lymphocyte trapping Changes in cell membraneProtection of antigen against enzyme degradationStimulation of macrophages and other cellsIsotype shifting Effect on immune regulators and several other immune responses.

Action at the level of antigen/protein molecule

Delivery vehicle or inert carrierAddition of the antigenic quality and hydrophobic groupConformational changes/change in the net electrical charges of the protein molecule

Action at the level of host

Action adjuvant

Increasing phagocytosis Levamisole, FK-565, MDP, LPS, FCA, Chitin,

Chitosan, Peptidoglycan, Vitamin C, ᵦ - Glucan

Increasing complement level ᵦ - Glucan, Vitamin C,

Increasing antibody production

FIA, aluminium salts, FK-565, LPS, Glucan

Increasing lysozyme production

Glucan, Levamisole, schizophyllan

Increasing leucocyte migration Quill A, saponin, glucan

Increasing interleukin-1 production

LPS

Effective action of some adjuvants

Adjuvant Types

Immunostimulants- levamisol, ᵦ-glucan, LPS

Carrier- oil imulsions, liposomes, micro and nanoparticles.

Surfactants- saponins

Particulates- aluminum salts

Conjugates- Link antigens to carriers

Biological- HSP, IFN, Ils, CpG

Adjuvants, Immunostimulants, and vaccine carriers used routinely in animal and human.

Reservoir and depots:

Latex beadsBentoniteSheep red blood cells(SRBC)Dimethyl sulphoxide(DMSO)

Carriers and vehicles:

Freund’s complete adjuvant(FCA)Freund’s incomplete adjuvant(FIA)LiposomesMineral oilDextran sulfate

B cell stimulators:

Inflammatory agents:Silica particlesCarbon particles

T cell stimulator:

Muramyl dipeptide (MDP)Glucan (yeast extracts)Aluminum saltsLevamisoleBCG

Lipopolysaccharides

Nutritional factors:Vitamin CVitamin K

Cytokines:InterlukinsInterferons

Polynucleotides:

CpG nucleotides

Cell membrane modifiers:

Saponins(plant extracts)Quill A

Different adjuvants creates its own type of immune responses but have some side effects.Aluminum adjuvants has inability to elicit cell mediated immune responses such as cytotoxic T cell responsesGranuloma formations and local inflammations in oil adjuvantsFreund’s complete adjuvant contain Mycobacterium : too harmful to be used as an adjuvant for human vaccines and restricted in certain countries.

To avoid this, different particulate (micro and nanoparticle based) adjuvants or carriers are better

alternatives.

Drawbacks of several adjuvants:

Vaccine or antigen or protein carriers are vehicles or transporters for a vaccine

orAn non-immunogenic molecule to which a hapten is bound

To enhance the immune response weak antigens.Maximum protection with less number of administration.Protect from acidic degradation in the peritoneum in oral delivery.Induction of more prolonged immune responses with controlled release of antigen

Liposomes

Liposomes are microscopic spherical vesicles composed of a phospholipid bilayers that are capable of encapsulating antigens.

Hydrophilic

Hydrophobic

Depending on the solubility of the antigen, it can be...

Encapsulated in aqueous core

Hydrophilic antigens incorporated into liposome

Taken up by bilayer

Diagram representinga liposome

encapsulating an

Antigen/vaccine in its

aqueous core.

Transmission electron micrograph of ECPs encapsulated chitosan coated liposomes (bar = 1.2 µm).

Antigen incorporated in chitosan coated lecithin based liposomes induces protective immune responses upon parenteral immunization

Natural Protein : gelatin, albumin, Collagen, Polysaccharides : alginate, dextran, Chitin, chitosan.Lipids: Lecithin, liposomesDisadvantages

Poor batch-to-batch reproducibilitySpecific condition for the degradationPotential antigenicity

Synthetic Polyesters : Poly D, L-lactic-Co-glycolic acid(PLGA), poly(ε-caprolactone) (PECL), PLA, Poly(alkylcyanoacrylate) (PACA), Polylactide(PLA), Polytetraflourothylene(PTFE), Silica, calcium phosphate etc.

Examples of biodegradable carriers: Alginate, chitosan, dextran, gelatin. PLGA, PLA, poly(acrylcyanoacrylate), PECL(Poly(έ-caprolactone)

Microparticles from natural or synthetic compound mostly microsphere and microcapsules Antigens coated with microparticles by covalent or non-covalent linkage and by physical entrapments.

Advantages:fewer antigen is required more efficiently presented to antigen presenting cellsless number of dose is required.

Scanning electron micrograph of outer membrane proteins loaded PLGA microparticles (bar = 1 mm).

The structure and properties of microparticles can be changed markedly with slight alternations in production conditions, to elicit significant change in immune responses.To avoid this sub micron particles, also called as nanoparticles can be used.It can reach the target organ more efficiently than the microparticles.By this we can change the surface charge and polarity

TEM micrographs of antigen/protein absorbed on the surface of calcium phosphate nano particles

Our Publications in this area T. Behera, P. K. Nanda, C. Mohanty, D. Mohapatra, P. Swain , B. K. Das, P. Routray, B. K. Mishra and S.K Sahoo, (2009). Poly D, L-lactide-co-glycolic acid (PLGA) microparticle encapsulated antigen stimulates both acquired and innate immune parameters through parenteral immunization in fish. Fish and shellfish immunology, 28:320-325.P. Swain, T. Behera , D. Mohapatra , P. K. Nanda, S. K. Nayak and P. K. Meher (2010). Derivation of rough attenuated variants from smooth virulent Aeromonas hydrophila and their immunogenicity in fish. Vaccine, 28:4626-4631.T. Behera, P. Swain, Antigen encapsulated in chitosan coated liposomes enhances immune responses through parenteral immunization. International Immunopharmacology, 2010 (In Press). S. K. Nayak, P. Swain, S. C. Mukherjee (2007). Effect of dietary suppliment of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.). Fish Shellfish Immunol.. 23: 892-896. P.Swain, S.K. Nayak, P.K. Nanda and S.Dash (2008). Biological Effects of Endotoxin in Fish- A Review. Fish and Shellfish Immunology, 25:191-2001Books Written:P. Swain, P. K. Sahoo and S. Ayyappan (2005). Fish and Shellfish Immunology: An Introduction, published by Narendra publishers, New Delhi, (ISBN 81-85375-90-9).

Thank you for

your Attention