VACCINE DELIVERY

BY

SAHITHI GADDEDEPARTMENT OF PHARMACEUTICSKVSR SIDDHARTHA COLLEGE OF

PHARMACEUTICAL SCIENCES VIJAYAWADA-10

Contents…..• About vaccines…• Mechanism of uptake and transport of

antigens…• Delivery systems used to promote uptake…

Lipid carrier systemsOral immunizationControlled release micro particles for vaccine

developmentSingle dose vaccine delivery systems using

biodegradable polymers..

• Peptide based and nucleic acid based vaccines…

History of vaccines……

VACCINE- from the Latin vacca (cow) The diary workers would never have the often fatal disease smallpox because they already have the cowpox

Jenner took the pus from the hand of a milkmaid with cow pox, scratched it into the arm of an 8 yr old boy and six weeks later

inoculated (variolated) the boy with small pox, he observed that boy did not catch smallpox.

History…….

Edward Jenner ( 1976)

The terms vaccine and vaccination are derived from variolae vaccinae (small pox of the cow), the term devised by Edward Jenner to denote cow pox.

Louis Pasteur ( 1880)

The second generation of vaccines was introduced in 1880s by Louis Pasteur who developed vaccines for chicken cholera and anthrax.

History ……

What are vaccines…????

• A vaccine is a biological preparation that improves immunity to a particular disease.

• A vaccine typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins.

• The agent stimulates the body’s immune system to recognize the agent as foreign, destroy it, and keep a record of it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

• Vaccines can be prophylactic ( to prevent or ameliorate the effects of a future infection by any natural or wild pathogen), or therapeutic ( vaccines against cancer).

Types of vaccines…

Vaccines are dead or inactivated organisms or purified products derived from them. The different

types of vaccines are:

a)Traditional vaccinesb)Innovative vaccines

a) Traditional vaccines

1. Killed -

2. Live, attenuated –

3. Toxoid -

4. Subunit vaccines- rather than introducing an inactivated or attenuated micro-organism to an immune system a fragment of it can create an immune response

b) Innovative vaccines1)Conjugate vaccines -

2) Recombinant vector vaccines (DNA) vaccines)

3) T-cell receptor peptide vaccines - they show the modulation of cytokine production and improve cell mediated immunity and are under development.

4) Valencei. Monovalent (univalent) - used to immunize against

single antigen. ii. Multivalent (polyvalent) - used to immunize

against two or more micro organisms.

5) Heterotypic – vaccines that are pathogens of other animals that either do not cause disease or cause mild disease in the organism being treated.

Mechanism of uptake and transport of antigens……

orhow do vaccines work..??

Y

Eliminated by:Killing of infected cells by CTL that use antigens generated by ENDOGENOUS PROCESSING

YEliminated by:Antibodies and phagocyteactivation by T helper cells that use antigens generated byEXOGENOUS PROCESSING

Antigens generated by endogenous and exogenous antigen processing activate

different effector functions

ENDOGENOUS PATHOGENS

EXOGENOUSPATHOGENS

Stages of exogenous antigen processing

UPTAKEAccess of native antigens and pathogens to

intracellular pathways of degradation

DEGRADATIONLimited proteolysis of antigens to peptides

ANTIGEN-MHC COMPLEX FORMATIONLoading of peptides onto MHC molecules

ANTIGEN PRESENTATIONTransport and expression of peptide-MHC complexes

on the surface of cells for recognition by T cells

Uptake of exogenous antigens

Y Y

Pinocytosis

Phagocytosis

Membrane Igreceptor mediateduptake

Y

Complement receptormediated phagocytosis Y

Fc receptor mediated phagocytosis

Uptake mechanisms direct antigen into intracellular vesicles for exogenous antigen processing

Endogenous antigen processing

UPTAKEAntigens/pathogens already present in cell

DEGRADATIONAntigens synthesised in the cytoplasm undergo limited proteolytic degradation in the cytoplasm.

ANTIGEN-MHC COMPLEX FORMATIONLoading of peptide antigens onto MHC class I

molecules is different to the loading of MHC class II molecules

PRESENTATIONTransport and expression of antigen-MHC complexes

on the surface of cells for recognition by T cells

ER membrane

Lumen of ER

Cytosol

Transporters associated with antigen processing (TAP1 & 2)

Transporter has preference for >8 amino acid peptides with hydrophobic C termini.

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

PeptideTAP-1 TAP-2

Peptide

TAP-1 TAP-2

PeptideTAP-1 TAP-2

Peptide

TAP-1 TAP-2

PeptideTAP-1 TAP-2

Peptide

TAP-1 TAP-2

PeptideTAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

ER membrane

Lumen of ER

Cytosol

TAP-1 TAP-2

Peptide

ATP-binding cassette(ABC) domain

Hydrophobic transmembranedomain

Peptide antigensfrom proteasome

Endoplasmic reticulum

TAP-1 TAP-2

Peptide

Cytoplasmic peptides are loaded onto the MHC molecule and the structure becomes compact

Cell surfacepeptidesof Ag

Antigens must be processed in order to be recognised by T cells

YT

T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

Solublenative Ag

Cell surfacenative Ag

Soluble peptidesof Ag

Cell surface peptides of Ag presented by cells that express MHC antigens

ANTIGENPROCESSING

Immune System

Myeloid Cells

Granulocytic

NeutrophilsBasophils

Eosinophils

Monocytic

MacrophagesKupffer cells

Dendritic cells

Lymphoid Cells

T cells

Helper cellsSuppressor

cellsCytotoxic

cells

B cells

Plasma cells

Natural Killer cells

How do vaccines work….

Response to vaccines…

Response to pathogens…

Are vaccines effective in all cases..??

The efficacy or performance of vaccine is dependant on a number of factors:

The disease itself The strain of vaccine Whether one kept to the timetable for

vaccinations Some individuals are not responders to

certain vaccines, meaning that they do not generate antibodies even after being vaccinated correctly

Other factors such as ethnicity, age, or genetic pre disposition.

Adverse effects

• Adverse effects if any are mild. • The rate of side effect depends on

the vaccine in question.• Some potential side effects include

FeverPain around the injection siteMuscle aches

Delivery systems used to promote uptake…..

Absorption enhancers: The term absorption enhancer usually refers to an

agent whose function is to increase absorption by enhancing membrane permeation, rather than increasing solubility, so such agents are sometimes more specifically termed as permeation enhancers.

Absorption enhancers are functional excipients included in formulations to improve the absorption of a pharmacologically active drug.

Ex: skin permeation enhancers include non-ionic surfactants which cause changes in the intracellular proteins of stratum corneum and increase permeability by this mechanism.

Lipid carrier systems

Liposome's are concentric bilayered vesicles in which hydrophilic moieties are enclosed by a membranous lipid bilayer mainly composed by natural or synthetic phospho-lipids

Role of liposome's in delivering vaccines.....

Liposome with antigens…

Uptake of the liposome by the cell membrane…..

Liposome's targeting to tumor cells

Viruses, proteins, glyco proteins, nucleic acids, carbohydrates and lipids can be entrapped and targeted at cellular and sub cellular level.

The development of polymerized liposome's which have shown enhanced stability in the GIT also offers potential for use in mucosal delivery.

Liposomal vaccines based on viral membrane proteins (virosomes) have been approved as products in Europe for hepatitis A and influenza.

Virosomes….

A virosome is a drug or vaccine delivery mechanism consisting of unilamellar phospholipid membrane (either a mono- or bi-layer) vesicle incorporating virus derived proteins to allow the virosomes to fuse with target cells.

Oral immunization

Most currently available vaccines are delivered by injection, which makes mass immunization more costly and less safe, particularly in resource-poor developing countries.

Oral vaccines have several attractive features compared with parenteral vaccines, but these are regarded historically as likely to be less effective, as vaccine antigens undergo digestion in the GI tract prior to induction of an immune response.

At present there are limited number of oral vaccines approved for human use, but many more are in the late stages of clinical development.

Due to the limited absorption from the intestinal tract and sensitivity to degradation, oral vaccines composed of killed bacteria and viruses or antigens isolated from infectious agents have not been successful.

New, live-attenuated bacterial and viral or edible plant-derived vaccines, how ever, have been recently introduced for this purpose.

Furthermore, systemic immunization with vaccines composed of bacterial polysaccharides chemically coupled to suitable protein carrier induces high levels of IgG antibodies, which may provide immunity toward Salmonella typhi, Shigella, and Escherichia coli.

Orally delivered vaccines are processed and presented by the digestive tract’s immune system, often referred to as the gut-associated lymphoid tissue (GALT).

The GALT is a complex system consisting of inductive sites ( where antigens are encountered and responses are initiated) and effector sites ( where local immune response occur) linked by a homing system, where by cells induced by antigen in the GALT migrate to the circulation and, subsequently colonize the mucosa.

As a result, oral vaccination can induce immune responses locally in the gut and at distant mucosal sites, as well as systemic humoral and cellular immune responses.

Oral vaccination typically generates a large amount of secretary IgA, which plays a major role in mucosal defense.

How oral vaccines induce immune responses…

Controlled release micro particles for vaccine development…

PLGA( polylactide co-glycolic acid) is used as a biodegradable micro particle for vaccine delivery due to the abundance of data and information on its properties, uses and role in on going studies.

A particular interesting area is the use of the biodegradable micro particles is to deliver DNA vaccines.

These are the third generation vaccines, and are made up of a small, circular piece of bacterial DNA ( called plasmid) that has been genetically engineered to produce one or two specific proteins ( antigens) from a pathogen

Preparation of PLGA micro particles………

Mechanism of release from microspheres is by bulk erosion.

Factors that effect the release pattern are:– Molecular weight of compound- greater the

mol. Wt. greater the bond, larger time to degrade.– Chemical composition of co-polymer- release

of the peptide was prolonged when microspheres made of copolymer containing higher proportion of polylactide.

– Size of the microspheres- greater the particle size longer the time to collapse, delays the release of antigen.

Biodegradable nano particles

Malaria is a mosquito borne disease caused by a parasite.Recent studies showed that upon encapsulating a subunit malarial antigen SPf66 in PLGA- mixture microspheres resulted in high antibody levels in mice.

Recent trends……..

Microencapsulation of vaccine antigen using biodegradable polymers seems a promising technology for needle-free targeted vaccination.

Single dose vaccine delivery systems using bio degradable polymers……

Single dose vaccines are given at a single contact point for preventing 4 to 6 diseases.

They would replace the need for a prime boost regimen, consequently eliminating the repeated visits to the doctor’s for mother’s and their children.

Disadvantage:– Cost compared to the current vaccine.

Use of biodegradable polymers….

Biodegradable Polymers: it comprised of monomers linked to one another through functional groups and have unstable links in the backbone.

These are broken down into biologically acceptable molecules that are metabolized and removed from the body via normal metabolic pathways.

Types of biodegradable polymers:

The two types are:• Natural biodegradable polymers

Ex: albumin, collagen, gelatin.

• Synthetic biodegradable polymersEx: aliphatic poly esters, poly anhydride,

poly ortho esters, pseudo poly amino acids etc.

Poly( lactide-o-glycolic acids) (PLGA) is most commonly used for vaccine delivery i.e. for preparation of microspheres.

Pre-filled syringes

Advantages :ConvenienceAffordabilityAccuracySterilitySafety

Preparation of pre-filled syringes

The steps include:• Preparation of container and closure

assembly of pre-filled syringes - mainly used are glass and plastic.

• Filling process in pre-filled syringes - bubble-free filling

• Sterilization of pre-filled syringes - by autoclave and ionizing radiations mainly by ionizing radiations that to gamma radiation

Peptide based vaccines….

A peptide vaccine is a type of subunit vaccine in which a peptide of the original pathogen is used to immunize an organism.

These types of vaccines are usually rapidly degraded once injected into the body, unless they are bound to a carrier molecule such as a fusion protein.

Delivery of peptides…

Antibodies usually bind to surface proteins of the pathogen or proteins generated after the disruption of the pathogen.Binding of antibodies to these proteins will stimulate an immune response.Therefore proteins can be use to stimulate an immune response.

Peptide based cancer vaccines

Nucleic acid based vaccines……

The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines

Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native confirmation, glucosylation profiles, and other post-translational modifications that mimic antigen produced during natural viral infection.

Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocytes responses to diverse protein antigens.

ADVANTAGES: Simplicity of the vector The ease of delivery Duration of expression

Nucleic acid vaccines are still experimental, and have been applied to a number of viral, bacterial and parasitic models of disease as well as to several tumor models.

Types of nucleic acids:1) DNA ( deoxy ribose nucleic acid) – contains the

genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). These segments carrying the genetic information are called genes.

2) RNA ( ribo nucleic acid) – it functions in converting genetic information from genes into the amino acid sequences of protein.

Direct DNA delivery in vivo can be utilized for the production of proteins as well as for the induction of specific cellular and humoral immune response against a large number of viral pathogens ( influenza, hepatitis b, HIV, etc.).

DNA vaccines… DNA vaccination is a technique for protecting an

organism against disease by injecting it with genetically engineered DNA to produce an immunological response.

These are the third generation vaccines, and are made up of a small, circular piece of bacterial DNA ( called plasmid) that has been genetically engineered to produce one or two specific proteins ( antigens) from a pathogen.

In 1996, trails involving T-cell lymphoma, influenza and herpes simplex virus were started.

How DNA vaccine is made…

Viral gene

Expression plasmid

Plasmid with foreign gene

Transform into bacterial cell

Plasmid DNA

Plasmid DNA get amplified

Plasmid DNA purified

Ready to use

Methods of deliveryMethod of Delivery

Formulation of DNA

Target Tissue Amount of DNA

Injection (hypodermic needle)

Aqueous solution in saline

IM (skeletal); ID; (IV, subcutaneous and intraperitoneal with variable success)

Large amounts (approximately 100-200 μg)

Gene Gun DNA-coated gold beads

ED (abdominal skin); vaginal mucosa; surgically exposed muscle and other organs

Small amounts (as little as 16 ng)

Pneumatic (Jet) Injection

Aqueous solution EDVery high (as much as 300 μg)

Topical application Aqueous solution Ocular; intravaginalSmall amounts (up to 100 μg)

Cytofectin-mediated

Liposomes (cationic); microspheres; recombinant adenovirus vectors; attenuated Shigella vector; aerosolized cationic lipid formulations

IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes

variable

Gene gun delivery

Pneumatic jet injection

Hypodermic needle injection

How DNA vaccines work….

DNA vaccine against cancer..

RNA vaccines…. Recent studies have demonstrated that

mRNA formulated in liposome's and administered sub-cutaneously or intravenously, effectively generated antibody and CTL’s directed against the encoded protein.

However, the difficulty and expenses of large scale RNA production and the relative instability of mRNA compared to DNA might render RNA vaccines an impractical means of immunization.

Conclusion…

Although various vaccines have been successfully developed for several diseases, research is still in progress to develop vaccines for life threatening diseases like cancer, AIDS etc..

Thank you…