Advance vaccine adjuvant

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Recent advances in Vaccine Adjuvants Dr P. Swain, Senior scientist, Fish Health Management Division Central Institute of Freshwater Aquaculture, Bhubaneswar .

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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”

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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 response Sometimes it gets the body to do something different and better then if it were naturally infected

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We recognize them as something different not belonging inside the body Once recognized we try and kill them We have two systems of doing this: The Innate immune system The adaptive immune system 2. The Battle Between Us and pathogens: What we can do How Vaccines Work

Vertebrates Immune System :

Vertebrates Immune System Thymus Mucosal associated lymphoid tissues(MALT) IgM antibody Little evidence of immunological memory Lack of bone marrow and lymph node Melano macrophage centres Thymus Haematopoitic tissues in bone marrow IgM amd IgG Evidence immunological memory Amphibia Fish

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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

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Birds Thymus Well defined Bursa fabricius- developes early Rapid memmory response IgM, IgG and IgA antibody Allergic response involving an IgE sub class Mammals Thymus Bone marrow for B cell Greater diversity in immunoglobulin Memory Allergic response involving an IgE sub class

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Immunoglobulin evolution

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Types of vaccine 1. Live, Attenuated (weakened) Vaccines A. First generation vaccines 2. Inactivated (killed) Vaccines

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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).

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C. Third generation vaccines DNA vaccines Peptide (synthetic) vaccines

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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..."

Many animal infectious diseases are also zoonotic and even cause massive loss to animal production :

Many animal infectious diseases are also zoonotic and even cause massive loss to animal production

An ideal Vaccine should be ….:

An ideal Vaccine should be …. 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

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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 unstable Degradation by the host enzymatic systems Therefore, the search for harmless and effective adjuvants is in process

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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

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

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How do adjuvants work? Antigen depot formation Lymphocyte trapping Changes in cell membrane Protection of antigen against enzyme degradation Stimulation of macrophages and other cells Isotype shifting Effect on immune regulators and several other immune responses. Action at the level of antigen/protein molecule Delivery vehicle or inert carrier Addition of the antigenic quality and hydrophobic group Conformational changes/change in the net electrical charges of the protein molecule Action at the level of host

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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

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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

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Adjuvants, Immunostimulants, and vaccine carriers used routinely in animal and human. Reservoir and depots : Latex beads Bentonite Sheep red blood cells(SRBC) Dimethyl sulphoxide(DMSO) Carriers and vehicles: Freund’s complete adjuvant(FCA) Freund’s incomplete adjuvant(FIA) Liposomes Mineral oil Dextran sulfate

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B cell stimulators: Inflammatory agents: Silica particles Carbon particles T cell stimulator: Muramyl dipeptide (MDP) Glucan (yeast extracts) Aluminum salts Levamisole BCG Lipopolysaccharides Nutritional factors: Vitamin C Vitamin K

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Cytokines: Interlukins Interferons Polynucleotides: CpG nucleotides Cell membrane modifiers: Saponins(plant extracts) Quill A

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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 responses Granuloma formations and local inflammations in oil adjuvants Freund’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 carriers:

Vaccine carriers Vaccine or antigen or protein carriers are vehicles or transporters for a vaccine or An non-immunogenic molecule to which a hapten is bound

Why vaccine/antigen carriers are used ?:

Why vaccine/antigen carriers are used ? 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

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

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Depending on the solubility of the antigen, it can be... Encapsulated in aqueous core Hydrophilic antigens incorporated into liposome Taken up by bilayer

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Diagram representing a liposome encapsulating an Antigen/vaccine in its aqueous core.

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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

Types of antigen/vaccine carriers used in medical and veterinary vaccines:

Types of antigen/vaccine carriers used in medical and veterinary vaccines Natural Protein : gelatin, albumin, Collagen, Polysaccharides : alginate, dextran, Chitin, chitosan. Lipids: Lecithin, liposomes Disadvantages Poor batch-to-batch reproducibility Specific condition for the degradation Potential 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.

Biodegradable carriers:

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

Micro particulate adjuvants or carriers:

Micro particulate adjuvants or carriers 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 cells less number of dose is required.

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

Nano particulate adjuvants or carriers:

Nano particulate adjuvants or carriers 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

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

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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, 25:191-2001 Books 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).

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Thank you for your Attention