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CONTROLLED DELIVERY OF VACCINE CONTROLLED DELIVERY OF VACCINE USING BIODEGRABLE SUBSTANCE

ORIGIN OF VACCINE Smallpox was the first disease which are inoculated with other type of type infections which was believed to be started in India and China before 200BC  Scientists who are involved are 

1) Lady Mary Worltey Montague 2) Edward Jenner 3) Sarah Nelmes 4) James Phipps

DEVELOPMENT OF NEW VACCINE

1998 first vaccine for rotavirus was developed; 21st century in 2006 human papillomavirus was developed for cancer.

DEFINATION OF VACCINE 

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Vaccine which are suspensions of killed, lived, or attenuated (having weakened virulence) cultures of micro-organism are used as antigen to produce immunity against infection due to the particular microorganism Example→ typhoid fever vaccine consists of killed cell salmonella typhii CHOLREA

TYPE OF VACCINE INACTIVATED VACCINE- Microorganism that has been killed chemicals or heat. This type of vaccine has less immune response due to which booster doses require.  Example – vaccine against flu, cholera 2) Live attenuated – These are live microorganism that has been cultivated under condition which disables their virulent properties. They have durable immunological reponse Example →yellow fever, measles, rubella and mumps 

CLASSIFICATION OF COMMON USED VACCINE DISEASE Tuberculosis

TYPE OF VACCINE Attenuated

Cholera

Inactivated

Polio

Attenuated

Influenza

Inactivated

Diphtheria

Inactivated exotoxin H influenza type Polysaccharide+

IMMUNIZATION SCHEDULE FOR CHILDREN IN INDIA AGE OF CHILD

VACCINE

1& ½ 2&½

BCG, DPT*1 AND PILIO*1 DPT*2 & PILIO*2

3&½

DPT*3 & PILIO*3

9 months

Measles

Below 16 and 24 months

DPT*4 & PILIO*4

DEVELOPMENT AND DESIGNING OF NEW GENERATION VACCINE Multivalent subunit vaccine (a)Small matrix antibody antigen complexes (b) Liposomes 2)Purified macromolecules 3)Synthetic peptides as vaccine 4) Immunoadhesions 1)

5) Antigen vaccine (a) Recombinant vaccines 6) Vector vaccines (d) Recombinant vector vaccine 7) Anti idiotype vaccines 8) Targeted immune stimulants

OBJECTIVE OF THE VACCINE DELIVERY To elicit a protective immune response of sufficiently long duration, from a singlecontact immunization  Potentiate the immune response to vaccine without manifesting any adverse effects on the body  Incorporate many in a single formulation deliver even those vaccines through the oral route that currently need to be given parenterally. 

INDUCTION OF IMMUNE RESPONSE Vaccine is carried out to protect the individual from by priming the immune system to resist the infecting agent.  This resistance can be offered by effectors molecules antibodies, cytokines, complement etc or effectors cell (cell mediated immunity).  Immunization also results in the system developing a “memory” of exposure to antigen present on the pathogen 

HOW IMMUNE RESPONSE IS GENERATED ? 

Immune response 1) Cell type immune response a) Non specific cell type i) Phagocytes ii) Auxiliary cell b) Targeted specific cell type i) B cell ii) T cell 2) Effector response

MECHANISM ACTION OF THE CELL TYPE IMMUNE RESPONSE 

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Phagocytes and auxiliary are first line defence against infection Inflammatory substance released from auxiliary cell on encounter with the foreigen material, stimulate phagocytes and attract from the site of infection. These cell get engulf the material upon the phagocytes Fragments of proteins produce are essential for generation of a T cell While performing the function macrophage act as antigen presenting cell (APC) The carry metabolic organelles to kill microorganism by generating the free radical

MECHANISM OF IMMUNOGENCITY BY CARRIER MOLECULES  The coupling of small peptides to carrier proteins

increase the molecular mass of peptide and there by improves the uptake by APC

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Biological half of small peptides may be increased via the linkage to carrier protein

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If the synthetic peptides itself is immunogenic (as in the case of peptides having B cell and T cell epitopes ), the carriers proteins merely functions as a polymeric delivery system.

MECHANISM OF IMMUNOGENICITY VIE CARRIER MOLECULES  2)

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Commonly used carrier are Keyholes limpet hemocyin (KLH) and sperm whale myoglobin (SWM) Albumins (ovalbumins) Fig will show the attachment of hapten to carrier molecules

EFFECTOR RESPONSE Effectors response of the T cell consist of secreting specialized substance called cytokines.  Cytokines are divided into two broad class 3) Helper T cell help B & T cell in variety of ways. 4) Cytokines or “killer” T cell make the cytokines that killed the infected cells Antibodies produce during this process are help in the in binding with invading microorganism and facilitating the pathogens bearing the Ag phagocytes. T cell are divided into TH1 which are primary involved in cell mediated immunity and TH 2 with the production of antibodies 

►T cell are divided into TH1 which are primary in cell mediated and TH 2 with the production ►TH 1 secrete gamma interferon TNF ►TH2 secrete IL-2, IL-5,IL-6, IL-10

CELL MEDIATED IMMUNITY On the exposure to the appropriate antigen T lymphocytes of lymphoid tissue proliferated to form activated T cell which are of three type 1) Helper T cell – release lymphokines which stimulate growth and differentiate of the activated B cell to from plasma cell 2)Cytotoxic T cell which bind tightly to specific binding antigen which then form secrete pore and form round role in the membrane 3) Suppresser T cell tthey are believed to inhibit the conversion of B cell into plasma cell 

DESIGING RATIONALE AND REQUIRMENT OF AN EFFICIENT VACCINE DELIVERY

 Particulate

Antigen  Booster Doses  Adjuvant – commonly used is Calcium phosphate ● Antigen Stability

PARTICULATE ANTIGEN 

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Uptake is generally take by phagocytes which are important for eliciting an immune response: the vaccine formulation should be amenable to phagocytosis. Soluble material which can be moved about anywhere is taken up by the pinocytosis. which is 10 times less efficiency than phagocytosis The efficiency of uptake can make the formulation in a particulate form. Finally particulate matter is important signal for phagocytic cells to home towards the site

BOOSTER DOSES Vaccine that are required to be given in the divided doses to produce the more efficiency  High doses tolerance is another curious phenomenon which occur if size of the doses is too high which rendered the antigen and no response is produced  Efficiency of the divide doses can be sought with the reference to cellular events that takes place in the immunization 

ADJUVANTS 

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Vaccine formulation is usually administered with the special additive called “adjuvant “ special when the vaccine is in the form of killed pathogens or isolated protein instead of live attenuated organism” “ A VACCINE DELIVERY SYSTEM IS ALSO EXPECTED TO HAVE SUFFICIENT ADJUVANTTICITY TO POTENTIATE THE REPONSE TO DELIVERY ANTIGEN.” Commonly used adjuvant are calcium phosphate which act as adjuvant and improved safety profile and enhance immune system stimulation

PRODUCTION OF CAP ♦The formulation of CAP nanoparticles is easily tailored for each antigen.  The manufacturing process is quick and requires only simple equipment, water for injection, and inorganic salts.  The CAP nanoparticle have exceptional protein loading capacity: about 20%(w/w) if antigen is coated on the surfaces only and about 50% and greater for internal “core loading”.  Batch-to batch consistency is excellent.  Long-term storage (out to one year) resulted in no changes in particles size, ph and surface morphology

ANTIGEN STABILITY Antigen stability – antigen used for immunization is subjected to environmental stress such as temperature ph or non-aqueous me3dium which may cause the change confirmation.  This may result the production of epitopes which are meant for generate the B cell response  Requirement for maintaining the stability of the antigen are follows – low temperatures but not freezing, physilogical ph and aqueous environment 

PREPARATION OF MICROSPHERE IN CONTROLLED DELIVERY  The properties for preparation of microsphere are 1) optimal antigen loading 2) size of microsphere 3) minimum wastage of material 4) within batch uniformity and inter-batch reproducibility 5) minimum exposures of Ag to denaturing condition 6) For parentrally products microsphere need to be sterile

POLY ( LACTIDE-CO-GLYCIDE) (PLGA) This type of the microsphere which is used for the controlled delivery system of peptides, native and synthetic proteins and lately.  PLGA microsphere are composed of a spherical shaped polymeric matrix ranging in diameter from 1 to 250 micrometere.  Many factors are important to formulate  i) Ability to release the entrapped substance ii) Particle size iii) Stability and safety which is related to in vivo polymer degration 

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Antigen are physically entrapped into microsphere inject able solid polymeric matrix The combination of diffusion through pores and of polymer matrix biodegradable allows the control of antigen release rates The biodegrading rates of polymer depends on its molecular weight After hydrolysis degraded products form monomer of lactide and glycolide which are eliminated by Krebs cycle as carbon dioxide & urine. During the process, the encapsulated antigen are release which can varies from hours to months depending upon the polymer combination

Microsphere contd  

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Microsphere diameter plays an important role in interaction with phagocytic cells Particle smaller than the 10 micrometer phagocytosed faster by the macrophage which cause recruitment to the site of administration after subcutaneous injection. Particle more than 10 micrometer act as depot releasing the antigen. Polymer selection also plays an important role in the manufactures because it critically influence their rate of biodegrading

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By changing the homopolymer ratio different physicochemical compositions can influence its degradability and permeability These parameter can be easily monitored using different polymer and copolymer Amorphous polymer are more permeable than PLGA The combination of particles with different compositions in the same formulation has permitted

MICROSPHERE PERPARATION This can be done by two methods 1) Simple emulsification (O\W) 2) Multiple water–in-oil-water (W\O\W) ♦ the choice of method depends on the physical and chemical characteristics of antigen, permitting the matrix entrapment of both lipophilic and hydrophilic ♦ in the first case an emulsion is formed by dissolving the antigen in an organic solvent immiscible in water (methlyene chlorides ) containing the polymer , under strong mechanical agitation . this emulsion can be stabilized by added surfactants to the aqueous phase. The solvent is eliminated by the evaporation at room temperatures, followed by the washed and freezing drying 

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The multiple emulsion method involves water-in-oil-inwater emulsification The inner aqueous phase containing the hydrophilic substance is obtained after emulsification with the immiscible organic solvent containing polymer under strong mechanical agitation. This emulsion is stabilized by the addition of aqueous solution containing a surfactants (e.g. poly vinyl alcohol ) and is further homogenized to produce a W\O\W double emulsion. This double emulsion is gently stirred with homogenizer room temperatures for solvent evaporation The microsphere are collected by the centrifugation, washed with water and freeze dried. the mixture of hydrphilic and lipophilic molecules can be successfully entrapped by this method

PGLA MICROSPHERE BIOCOMPATIBLITY Polymer microspheres have attracted much attention because of their biocompatible characteristics the phagocytosis of biodegradable and no biodegradable particles has reported to depend on their size, surface charge and hydrophobicity. ♦ After subcutaneous administration, PLGA microsphere ranging in the diameter from 1 to 10 micrometer is readily phagocytosed by macrophages recruited to the site of injection, thereby providing an intracellular delivery of the antigen. ♦This mechanism may enhance antibody response and consequently decreases the required antigen dose. On the other hand, particles larger than 10 micrometer in diameter would remain as a depot at the site of injection providing a sustained release of antigen.  Improving their hydrophobicity can increase phagocytosis of the particles, whereas microsphere preparation with different compositions of PLA and  PLGA do alter the extent of phagocytosis. 

ROUTE OF IMMUNIZATION The oral route of delivery is the most acceptable in terms of patient compliance.  the antigen entering the body through the oral route include items of food, and are “tolergenic”.  Lymphoid tissue associated with the digestive tract is located primarily in Peyer’s patches in the small intestine and the material that targets to Peyer’s patches often evokes an immune response.  This response is often in a such type as protect against pathogens entering the body by invading mucosal tissue if the response against microbes invading by non-mucosal routes is desired after oral immunization, the delivery system must be capable of transporting the Ag to deep sealed lymphoid tissue instead of releasing it to the mucosa-associated lymphoid tissue.  Systemic routes of immunization should seek a route that ensures maximal exposures of the Ag to immunocytes.  The usual choices are intramuscular (i.m.) subcutaneous or interdermal immunization. 

As th e site deliv ery mo ves fr om deep tis sues towa rd s su perfi cial, the d eli very syste m is e xpose d to a la rg er n umb er o f fi rst l in es defe nce cell (i. m, i.d ) at th e same time , the perio d o f re sid ence of the d eli very syste m in the b ody decrease a s the sit e o f deliv ery moves t owa rds su perfic ia l tiss ue . Route of immunization also takes place by two way 1) Systemic immunization 2) Mucosal immunization 

CONCLUSION PLGA microsphere with an incorporated antigen represents a good antigen delivery system for both cellular and humoral response.  The easy manufactures of microsphere and the possibility of administration by different routes offer the additional advantage of their use as a pharmaceutically acceptable adjuvant for vaccines. 

INSTRUMENTAL METHOD OF PERPARATION This method include 2) Spray drying 3) Air suspension coating 4) Press grinding 5) Coaceration-Phase separation i) Emulsification methods a) Oil-in-oil emulsion b) Oil-in-water emulsion c) Multiple emulsion 5) Solvent extraction 6) Rotary evaporations 

SPRAYING DRYING In this method spray dried protein is suspended in a solution of the polymer in an organic solvent (methylene chloride ortetrahydronfuran). 1)This is then pumped into spray drier by a peristaltic pump. 2) Dry air at high pressure and inlet air temperature of 37ºC atomizes the air suspension, and the polymer forms a matrix entrapping the protein as the solvent evaporates from the droplets.  Spherical particles of the desired size range and loading can thus prepared. 

DISADVANTAGE OF THIS METHOD  This method is relatively large batch size required for processing in view of the limitation of the availableequipment. ♦ Even small volumes spray driers are not designed to handle volumes of the order of a few ml, which would be sufficient to produce thousands of doses.  This size dispersion of microsphere produced by this method is also exposed to be large. 

EVALUATION OF VACCINE LAODED MICROSPHERE  The

evalulation factors involve 1)Praticle size 2) Antigen loading 3) Structural integrity of encapsulated antigen 4)Studies on the antigen release in vitro 5) Antigencity of the preparation

Praticle size 

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Particles size can be easily determine using light microscopy or in the greater detail by scanning the electron microscopy (SEM) which shows the scanning electron microphotography of abatch of the microsphere. SEM also helps in accessing surface morphology of the microsphere. Standards equipment for micromerities such as Coulter Counter or laser

Studies on the antigen release in vitro 

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In vitro Ag release studies have served as quality control parameters as well as index of the duration of immune response. These help to understand the nature of release and provide significant insight into the formulation variables By dispersing the microsphere in a suitable buffer in a vial and subjecting it to stirring at 37ºc, over the period of time.

NOVEL DRUG SYSTEM Novel drug system include following point 1)Particulate delivery system i) Liposome ii) Emulsion iii) Micro sphere 2)Cochleates 3)Mucoadhesive polymer 4)DNA vaccination 5)Transgenic plants an edible immunogen concept