This chapter contains concepts of vaccine delivery, antigens uptake, single shot vaccines, mucosal and transdermal delivery of vaccines.

1. VACCINE DELIVERY SYSTEMS
INTRODUCTION:
The word “vaccine” originates from the Latin Variolae vaccinea (cowpox), which Edward
Jenner demonstrated in 1798 could prevent smallpox in humans. Today the term ‘vaccine’
applies to all biological preparations, produced from living organisms, that enhance immunity
against disease and either prevent (prophylactic vaccines) or, in some cases, treat disease
(therapeutic vaccines).
Vaccines are administered in liquid form, either by injection, by oral, or by intranasal routes.
Ex. Polio , Hepatitis –A……etc
DEFINITION:
 Vaccines are biological preparations that provides active acquired immunity to a
particular disease.
 Vaccine typically contains a disease causing microorganism.
 It is often made from weakened or killed forms of the causative microbes or its toxins or
one of its surface proteins.
 The agent or product through which immunization is active are called immunization
agents.
 Vaccines may be single component or mixed component vaccines.
ADVANTAGES:
1. No disease-transmission risk .
2. No auto immunogenic or anaphylaxis.
3. Broadly applicable with almost all important drugs (anticancer drugs , proteins, peptides,
nucleic acids , antibiotics, fungicides).
4. Enables drug delivery into the cytoplasm of target cell.
5. Protects drugs against degradation.
DISADVANTAGES:
1. Shelf-life is too short.
2. Scale up related problems.
3. Poor quality of raw material.

2. TYPES OF VACCINES:
TYPE LIFE ATTENUATED
VACCINE
KILLED VACCINE
Bacterial
Viral
Rickettsial
Toxoids
Tuberculosis (BCG)
Small pox
Rubella
Measles
Yellow fever
Mumps
Diphtheria
Tetanus
Cholera
Typhoid
Whooping cough
Poliomyelitis
Influenza
Rabies
Typhus
MECHANISM OF VACCINE:

3. UPTAKE OF ANTIGENS:
 The components of the disease-causing organisms or the vaccine components that trigger
the immune response are known as “antigens”.
 These antigens trigger the production of “antibodies” by the immune system. Antibodies
bind to corresponding antigens and induce their destruction by other immune cells.
 The induced immune response to either a disease-causing organism or to a vaccine
configures the body’s immune cells to be capable of quickly recognizing, reacting to, and
subduing the relevant disease-causing organism.
 When the body’s immune system is subsequently exposed to a same disease-causing
organism, the immune system will contain and eliminate the infection before it can cause
harm to the body.
(1) Stages of Exogenous antigen uptake:
UPTAKE
Access of native antigens and pathogens to intracellular pathways of degradation
DEGRADATION
Limited proteolysis of antigens to peptides
ANTIGEN-MHC COMPLEX FORMATION
Loading of peptides onto MHC molecules
ANTIGEN PRESENTATION
Transport and expression of peptide-MHC complexes on the surface of cells for recognition
by T cells.

4. What is MHC ?
 The major histocompatibility complex (MHC) is a set of cell surface proteins that are
essential for the acquired immune system to recognize foreign molecules in
vertebrates, which in turn determines histocompatibility.
 The main function of MHC molecules is to bind to antigens derived from pathogens
and display them on the cell surface for recognition by the appropriate T cells.
(2) Stages of Endogenous antigen uptake:
UPTAKE
Antigens/pathogens already present in cell
DEGRADATION
Antigens synthesised in the cytoplasm undergo limited proteolytic degradation in the cytoplasm
ANTIGEN-MHC COMPLEX FORMATION
Loading of peptide antigens onto MHC class I molecules
is different to the loading of MHC class II molecules
PRESENTATION
Transport and expression of antigen-MHC complexes on the surface of cells for recognition by T
cells
SINGLE SHOT VACCINES:
 Single shot vaccines are given at a single contact point for preventing 4-6 diseases.
 They will replace the need for a prime boost regimen, consequently eliminating the
repeated visits to doctors.
 The cost for single shot vaccines are higher as compared to normal vaccines.
DEFINITION: The single shot vaccine is a combination product of a prime component antigen
with an microsphere component and appropriate adjuvant and an encapsulated antigen which
will provide the booster immunizations by delayed release of the antigen.
 In order to increase the therapeutic activity of single shot vaccines vaccine adjuvants are
used.
 Addition of adjuvants triggers the immune system to become more sensitive to vaccine.
Particulate adjuvants.
They form very small particles that can stimulate the immune system and also enhances the
delivery of antigen to immune cells.
Examples of particulate adjuvants are:
(1) ALUM
 Most commonly used adjuvant.
 Consists of Aluminum salts that are not soluble in water.
 Recently it is used in vaccines for Hepatitis B.
 Mechanism is unknown for how it stimulates vaccine induced immunity.

5. (2) VIROSOMES
 They are resemble to viruses but non-infectious.
 They are included in Flu vaccine and Hepatitis A vaccine in Europe.
 Virosomes that are incorporated in these vaccines have antigens and other proteins on
their surface, but they can not cause infection because it does not contain any genetic
material.
 Mechanism: some immune cells recognize these particulate cells and engulf them and
present them to immune system and mount protective response.
(3) CYTOKINES
 They are small proteins that serves as chemical messenger of the immune system.
 Because of their role in immune responses, some cytokines have been evaluated as
vaccine adjuvants.
 Sometimes these adjuvants are used in combinations for producing proportional immune
responses.
Important determinants for single shot vaccine development:
 Biodegradable technology
 Encapsulation Efficiency
 Particle size distribution
 Preservation of bio activity during formulation and released
 Scalable production processes
 Effects of combination with various adjuvant
 Effects of different administration route.
MUCOSAL VACCINE DELIVERY SYSTEM:
 Mucosal surfaces area is major portal of entry for many human pathogens that are the
cause of infectious diseases worldwide.
 Immunization by mucosal routes may be more effective at inducing protective immunity
against mucosal pathogens at their sites of entry.
 Efforts have focused on efficient delivery of vaccine antigens to mucosal sites that
facilitate uptake by local antigen-presenting cells to generate protective mucosal immune
responses.
 Discovery of safe and effective mucosal adjuvants are also being sought to enhance the
magnitude and quality of the protective immune response.
 The adult human mucosa lines the surfaces of the digestive, respiratory and genitourinary
tracts, covering an immense surface area (400m2) that is ~200 times greater than that of
the skin. It is estimated that 70% of infectious agents enter the host by mucosal routes.
 Mucosal surfaces are typically categorized as type-I and type-II mucosa. Type-I mucosa
include surface of the lung and gut, where as type-II mucosa include surfaces of the
mouth, esophagus and cornea.
 The female genital tract has both type-I and type-II mucosa.

6. Designand strategies for mucosal delivery.
(1) Emulsion type delivery
 Emulsions are heterogeneous liquid systems may be water-in-oil emulsions(w/o), oil in
water emulsions(o/w), or more complex systems such as water-in-oil-in-water(w/o/w)
multiple emulsions, micro emulsions or nano emulsions.
 Antigens are dissolved in a water phase and emulsified in the oil in the presence of an
appropriate emulsifier.
 The controlled release characteristics of an emulsion are determined by factors such as
viscosity of oil phase, oil-to-water phase ratio and emulsion droplet size.
E.g. High oil content cause unnecessary injection site irritation and too large a droplet size can
result in a physically unstable product there by reducing its shelf life.
 HUANG ET developed a novel emulsion-type vaccine delivery systems of the
amphiphilic bioresorbable polymer poly(ethylene glycol)-block-poly(lactide-co-epsilon-
carpolactone) (PEG-B-PLACL) using Ovalbumin as model antigen.
 Results from physiochemical characterization studies and in-vitro release studies showed
that they are composed of homogeneous fine particles.
Advantages: Slow release of antigen
Disadvantages: Access immunogenic response
Fever
Sore arm at injection site.
(2) Liposome based delivery
 Liposomes are spherical shape vesicles containing an aqueous core which is enclosed by
a lipid bi-layer.
 They are most often composed of phospholipids, especially phosphatidylcholine, but may
also include other lipids, such as eggphosphatidyl-ethanolamine.
Preparation of liposome vaccine delivery system.
Depending on the chemical properties, water-soluble antigens (proteins, peptides, nucleic acids,
carbohydrates, haptens) are entrapped within the aqueous inner space of liposomes.
Lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into
the lipid bilayer.
Antigens or adjuvants can be attached to the liposome surface either by adsorption or stable
chemical linking.
Liposome

7. Advantages: Easy surface modification
Synthesized from non-toxic material
Wide range of antigen encapsulation
Plasticity
Disadvantages: Stability problem
Low antigen loading. Nanoparticle
(3) Polymeric nanoparticles
 Polymeric nanoparticles (PNPs) are submicron-sized colloidal particles.
 Polymeric nanoparticles because of their size are preferentially taken up by the mucosa
associated lymphoid tissue.
 Limited doses of antigen are sufficient to induce effective immunization.
 Hence, the use of nanoparticles for oral delivery of antigens is suitable because of their
ability to release proteins and to protect them from enzymatic degradation in the GIT.
Biodegradable poly(alkyl cyano-acrylate) (PACA)
 Nanoparticles have been shown to enhance the secretory immune response after their oral
administration in association with Ovalbumin in rats.
Biodegradable poly(methyl metha acrylate) (PMMA)
 Nanoparticles being very slowly degradable (30%-40% per year) appear to be
particularly suitable for vaccine purposes because prolonged contact between antigen and
immunocompetent cells favors persistent immunity.
(4) Virosomes
 A virosome is a drug or vaccine delivery mechanism consisting of unilamellar
phospholipid membrane ~150mm (either a mono or bi-layer) vesicle incorporating virus
delivered proteins to allow the virosomes to fuse with target cells.
 These proteins enable the virosome membranes to fuse with cells of the immune system
and thus deliver the specific antigens directly to their target cells.
 They elicit a specific immune response even with weak immunogenic antigens.
 Once they have delivered the antigens, the virosomes are completely degraded within the
cells.
 A viral protein attached on the phospholipid bilayer not only confers structural stability
and homogeneity to virosomal formulations, which are clearly distinct from other
liposomal carrier systems.
 Virosomes represent vesicular systems into which antigens can be loaded into virosomes
or adsorbed onto the virosomal surface through hydrophobic interactions.

8. (5) Melt in mouth strips
 Quick dissolving films containing immunogens.
 Melts into liquid that children and infants will swallow easily.
 First designed by undergraduate students at johns Hopkins university on biomedical
engineering design day for protection against ROTA virus infection.
 ROTA virus is a common cause of severe diarrhea and vomiting in children, leading to
about 600000 deaths annually. ROTA virus vaccine at present is available in a liquid or
freeze-dried form that must be chilled for transport and storage, making it very expensive
for use in impoverished areas.
 In addition, newborns sometimes spit out the liquid, a problem that is less likely to occur
with a strip that sticks to and dissolves on the tongue in less than a minute.
TRANSDERMAL VACCINE DELIVERY SYSTEM:
 The skin is the largest and most accessible organ of the body.
 Vaccine administration to the skin offers many advantages including ease of access, a
potential for generation of both systemic and mucosal immune response.
 Formulation approaches such as liposomes, physical penetration enhancers such as
electroporation and technologies that create micron-sized pores in the skin such as
microneedles.
Skin as a site of vaccine delivery
The skin has multiple barrier properties to minimize water loss from the body and prevent
the permeation of environmental contaminants into the body.
These barriers can be considered as physical, enzymatic and immunological.
1) Physical barriers
The outermost layer, stratum corneum presents an effective physical barrier to the
permeation of large molecules such as vaccines. This is the first barrier property that must be
overcome to provide effective transdermal vaccine delivery.
2) Enzymatic barrier
The skin possesses many enzymes that are capable of hydrolyzing peptides and proteins.
Their potential to degrade topically applied vaccine antigens should be considered.

9. 3) Immunological barriers
When the skin is damaged, environmental contaminants can access the epidermis to initiate
an immunological response.
Many approaches have been investigated to overcome the skins barrier properties in order
to deliver antigens via the skin.
All methods aim to overcome the stratum corneum barrier and target vaccine to immune
responsive cells such as Langerhans cells.
Designand stratergy for transdermal vaccine delivery.
(1) Liquid jet injectors
Liquid jet injectors use a high-velocity jet (typically 100 to 200 m/s) to deliver molecules
through the skin into the subcutaneous or intramuscular region.
 Jet injectors can be broadly classified into,
a) Multi-use nozzle jet injectors (MUN JIS)
b) Disposable cartridge jet injectors (DC JIS)
 Commercially available liquid jet injectors consists of,
(A) Power source (compressed gas or spring)
(B) Piston
(C) Drug or vaccine-loaded compartment
(D) Application nozzle, with typical orifice size in the range of 150 to 300 μm.
Working of jet injectors
 Upon actuation the power source pushes the piston & rapidly increases the pressure
within the drug-loaded compartment.
 Thereby forcing the drug solution through the orifice as a high velocity liquid jet.
 When the jet impacts on the skin it creates a hole & allows the liquid to enter the skin.
 The process of hole formation and liquid jet deposition occurs within microseconds.
 The deposited liquid can then disperse within the tissues to illicit an immune response.
Applications of jet injectors.
1. Applications of liquid-jet injectors have been focused on delivery of macromolecules that do
not passively permeate the skin.
2. It has been shown to increase immune responses to both conventional and DNA-based
vaccines.
E.g. Hepatitis A vaccine or a influenza vaccine, were found to be increased by atleast 10% when
using needle-free injectors compared to needle and syringe administration.

10. Demerits of jet injection technology
1. Pain and bruising at the site of administration
2. Recently novel pulsed micro-jet system is designed to reduce the adverse effects at the site of
administration.
Electric transducer is used to control the delivery volumes, jet diameters (50-100 mm) and
injection velocity (>100 m/s) thus minimizing pain and tissue damage.
(2) Epidermal powder immunization
 Powder injectors were first used for DNA and RNA transfection into plants.
 The technique has subsequently been investigated for transdermal protein delivery, gene
therapy and vaccination.
 The device design principles are similar to liquid injectors, with a powder compartment
and compressed carrier gas, such as helium. Upon actuation, the particles are carried by
the gas, to impact the skin surface at high velocity, puncturing micron-sized holes in the
epidermis to facilitate skin deposition.
 Upon actuation, the particles are carried by the gas, to impact the skin surface at high
velocity, puncturing micron-sized holes in the epidermis to facilitate skin deposition.
Advantages.
1. Powder injectors offer advantages over liquids in terms of formulation and stability issues.
2. Initial safety studies suggest that the powder injectors are reasonably well tolerated, and the
particle bombardment offers advantages with regard to Langerhans cell targeting and immune
system activation.
(3) Colloidal carriers
 The rationale for the use of colloidal carriers is that compounds with unfavourable
permeation characteristics can be packaged within carriers that will permeate the skin.
 There has been considerable research in the application of liposomes and lipid particle
carriers, there is no conclusive evidence that these carriers can permeate the skin intact.

11. (A) Nanoparticle and Nano carriers
 Nanoparticles and microparticles are polymeric particles in the nanometer and
micrometer size range respectively.
 Compounds can be incorporated into the particles in form of a solid dispersion or a solid
solution, or bound to the particle surface by physical adsorption and chemical binding.
 Thus allowing the particles to act as carriers or as adjuvants for the vaccine.
 Problems associated;
 But the general problem is that nanoparticles administered to the skin do not permeate the
intact stratum corneum, but may accumulate in hair follicles. So their potential utility for
passive transdermal vaccine delivery is limited.
(B) Liposomes and elastic vesicles.
 Liposomes consists of multiple bilayers of phospholipids capable of solubilising both
lipophilic and hydrophilic compounds within their structure.
 They could act as skin permeation carriers.
 Evidence of their permeation across the stratum corneum intact has not emerged.
 But alteration of the composition including incorporation of surfactants will give elastic
or deformable liposomes which are capable of deforming in shape.
 So they could be “Squeeze through” narrow pores in the stratum corneum.
Limitations
Research area for the permeation enhancement of small molecules for vaccine development is
very limited due to the stability considerations with these systems.
(4) Energy based approaches
 Exposure of the skin to energy in the form of electrical pulses or ultrasonic waves can
disrupt the stratum corneum barrier and increases its permeability.
 This approach has been extensively investigated for drugs and macromolecules, and to a
lesser extent for vaccine delivery.
(A) Electroporation
 Electroporation involves the administration of electrical pulses to create transient pores in
the skin and thus increase the skin permeability to drugs and macromolecules.
 INOVIO Biomedical Corporation has developed a series of hand-held, cordless
electroporation devices that have been used in vaccine delivery studies
 Delivery of DNA vaccines into muscle or skin tissue with electroporation systems
generated robust immune responses.

12. (B) Ultrasound or sonophoresis
 Low frequency sonophoresis involves application of ultrasound waves at frequencies
between 20 to 100 kHz to the skin surface to reduce the stratum corneum barrier and
thereby increase skin permeability.
 Pretreatment is given prior to the application of a drug solution or patch.
 Low frequency ultrasound (20 kHz) was used to deliver a tetanus toxoid, eliciting a
robust immune response in mice to mice.
 A commercial ultrasound device, SONOPREP, for administration of local anesthetic, was
launched in 2004 but withdrawn in 2007.
(C) Thermal ablation or microporation
 Thermal ablation generates micron-size holes in the stratum corneum by selectively
heating small areas of the skin surface to hundreds of degrees.
 The heat is applied for micro-to milliseconds so that heat transfer to the viable tissues is
avoided, thus minimizing pain and damage.
(D) Microneedles
 Microneedles consists of pointed micro-sized projections, fabricated into arrays with up
to a hundred needles, that penetrate through the stratum corneum to create microscopic
holes, thus providing delivery pathways for vaccines and drugs.
A number of different microneedle systems have been investigated including:

13. a) Solid microneedles for permeabilizing skin via formation of micron-sized holes.
 Solid or insoluble micrneedles are generally composed of metal such as titanium or
silicone.
 The microneedles permeabilize the skin by forming micron-sized holes through the
stratum corneum.
 The microneedle ray is then removed and a drug/vaccine containing patch is applied. This
approach is termed “POKE & PATCH”.
b) Solid microneedles coated with dry drug or vaccine.
 Coated microneedles have an insoluble core coated with drug that dissolves off within the
skin.
 This is called “COAT & POKE” approach.
c) Polymeric microneedles with encapsulated drug or vaccine.
d) Hallow microneedles
 Insoluble hallow microneedles create holes through which the drug solution can pass into
the skin.
 Of these, the development of insoluble solid and hallow microneedles is most advanced
for vaccine delivery.
 Clinical studies generally report no significant adverse effects from microneedles,
including minimal erythema and pain, most likely because the projections are not long
enough to reach nerve endings in the deeper tissue.