Immunization

Immunity

Specific defensesImmunity

Passive immunityActive immunity

Following clinical infection

Following subclinical infection

Following vaccination Following administration ofImmunoglobulin or antiserum

Transfer of maternal Antibodies Through milk

Transfer of maternal Antibodies Through placenta

natural

acquired

Immunizing agents

Immunizing agents

antiseraimmunuglobulinsvaccines

Purpose of Vaccination

Protect the individual from disease.Reduce the severity of disease.Protect the community.Eradication of the disease.

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Reduce the burden of disease.

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Luis Fermín Tenorio,

the last polio case in the Americas

Peru, 1991

Ali Maouw Maalin,last case of

smallpox (Somalia,1977)

Vaccines are the most cost-effective tools for preventing death and disability from infectious disease

The value of VaccinesThe value of Vaccines

Basic concept of vaccines6

Deliver to the body some part or all of the disease organism that IMITATES the pathogen but is not pathogenic. Induce protective immune response.

Polysaccharide

Surface proteins

Intracellular proteins

Toxins

Entire organism• live (attenuated)• killed

LPS

capsular

Tools available to develop a Tools available to develop a processprocess

Growthof

Virus, Yeastor Bacteria

Clarification

Inactivation

PL

Formaldehyde

Eggs

Bacterial / Yeast

Fermentation

Cell culture

Depth filter

Crossflow filtration

Centrifuge

PurificationConcentration ++

Crossflow filtration

Chemicalprecipitatio

n

Ultracentrifugation

Chromatography

Ion exchangeHydrophobic

AffinitySize exclusion

Cross-flow filtration

The majority of the feed flow travels tangentially across the surface of the filter, rather than into the filter. The principal advantage of this is that the filter cake (which can blind the filter) is substantially washed away during the filtration process, increasing the length of time that a filter unit can be operational.

It can be a continuous process.

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Vaccine manufactureVaccine manufactureAntigen ProductionAntigen Production

EggsEggs– InfluenzaInfluenza

Bacterial / Yeast Bacterial / Yeast fermentationfermentation

– Whole organism (e.g. Cholera)Whole organism (e.g. Cholera)– Subunit vaccines (e.g. Capsular Subunit vaccines (e.g. Capsular

polysaccharide, Tetanus and polysaccharide, Tetanus and Diphtheria toxoid)Diphtheria toxoid)

– Genetically engineered proteins (e.g. Genetically engineered proteins (e.g. Hepatitis B and HPV vaccines)Hepatitis B and HPV vaccines)

Cell cultureCell culture– Viral vaccines either whole virus or Viral vaccines either whole virus or

subunitsubunit– Genetically engineered proteinsGenetically engineered proteins

FormulationAntigen

presentation

ISCOMS

ADJUVANTS

VIRUS LIKE PARTICLES

VIROSOMES

CONHNHCO(CH2)4CONHNHOC- DTVi-CPS

CONJUGATES Combining Combining antigen(s)antigen(s)

Combining with Combining with adjuvantadjuvant

StabilizersStabilizersPreservativesPreservatives

Cryo-protectantsCryo-protectants

Fill and Finish

A conjugate vaccine is created by covalently attaching a poor (polysaccharide organism) antigen to a carrier protein (preferably from the same microorganism).

Immune stimulating complexes (ISCOMs) are spherical open cage-like structures that are spontaneously formed when mixing together cholesterol, phospholipids and Quillaia saponins under a specific conditions.

the ISCOM-Matrix is simply mixed with the antigen post-manufacturing, the antigen can also be incorporated into the structure.

Adjuvant: The word “adjuvant” comes from the Latin word adiuvare, meaning to help or aid. "An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens."

Virosome: 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. Virosomes are not able to replicate but are pure fusion-active vesicles.

A cryoprotectant is a substance used to protect biological tissue from freezing damage (antifreezers).

Conventional cryoprotectants are glycols, such as ethylene glycol, propylene glycol, and glycerol.

Types of Vaccines13

Types of vaccines

Live vaccinesAttenuated live vaccinesInactivated (killed vaccines)ToxoidsPolysaccharide and polypeptide (cellular

fraction) vaccinesSurface antigen (recombinant) vaccines.

Types of vaccinesLivevaccines

LiveAttenuated vaccines

KilledInactivated vaccines

Toxoids Cellular fraction vaccines

Recombinant vaccines

•Small pox variola vaccine

•BCG•Typhoid oral•Plague•Oral polio•Yellow fever•Measles•Mumps•Rubella•IntranasalInfluenza•Typhus

•Typhoid•Cholera•Pertussis•Plague•Rabies•Salk polio•Intra-muscular influenza•Japanise encephalitis

•Diphtheria•Tetanus

•Meningococcal polysaccharide vaccine•Pneumococcal polysaccharide vaccine•Hepatitis B polypeptide vaccine

•Hepatitis B vaccine

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Inactivated Toxins

Exotoxin Gram negative and

gram positive bacteria.

Heat Labile. Protein. Secreted by the

bacteria.

Endotoxin Gram negative

bacteria. Heat stable. Lipopolysaccharide. Firmly bound to the

bacteria outer membrane.

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Toxoid

- Not a vaccine against the organism.- Inactivation of toxin by chemical

(formalin) or heat treatment.Vaccine against pathogenic exotoxinTetanus, diphtheria, (pertussis?), anthrax?

Purify toxin then chemically inactivate (toxoid) Risk of incomplete inactivation. TT (Tetanus toxoid), DT.

Genetically modify toxin so non-toxic CRM (diphtheria), mLT (cholera, ETEC)

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Whole Bacteria or Whole Bacteria or Virus VaccinesVirus Vaccines

Cholera bacteria Polio

virus

Live vaccines

Live vaccines are made from live infectious agents without any amendment.

The only live vaccine is “Variola” small pox vaccine, made of live vaccinia cow-pox virus (not variola virus) which is not pathogenic but antigenic, giving cross immunity for variola.

Subunit

Purify a protein or proteins from pathogen

Selective presentation of 'protective' antigens Pertussis

Pertussis toxin + filamentous haemagglutinin + pertactin (no LPS)

Influenza (subunit) Mainly haemaglutinin + neuraminidase

Disadvantages Requires growing the pathogen and purifying

protective (antigens) subunits.

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Bordetella pertussisBordetella pertussis

Filamentous haemagglutinin

Pertactin

Pertussis toxin a 69 kD outer membrane protein (OMP).

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Different forms of Polysaccharide

LPS Capsular polysaccharide

Polysaccharide

Many bacteria produce a strain-specific capsular polysaccharide on their surface.

Antibody to these antigens are protective. Streptococcus pneumoniae, Haemophilus Type

B, Typhoid (Vi). Can be easily purified.Immunogenic in older children / adults.But poorly immunogenic in infantsT-cell independent responses

Short lived Low antibody responses

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Conjugate Conjugate VaccinesVaccines

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NHNHCO(CH2)4CONHNH

Exoprotein A (rEPA)

SpacerC=O

C=O

Vi

Capsular Polysaccharide

Vi Conjugate vaccineVaccine against Typhoid Fever

Vector Vaccine

A vector vaccine is a vaccine which is introduced by a vector e.g. vaccinia virus.

The vaccinia virus as a live vaccine led to the globally eradication of the smallpox virus.

The genome of the vaccinia virus has been completely sequenced.

The vaccinia virus is generally nonpathogenic.

INTRODUCTIONINTRODUCTION

DNA vaccine is DNA sequence used as a vaccine.

This DNA Sequence code for antigenic protein of pathogen.

As this DNA inserted into cells it is translated to form antigenic protein. As this protein is foreign to cells , so immune response raised against this protein.

In this way ,DNA vaccine provide immunity against that pathogen.

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Recombinant VaccinesRecombinant Vaccines

S. cerevisiae

E.coli

30RecombinantProtein produced by genetic engineering

Express protective antigen in safe easy-to-grow organism Hepatitis B (HBsAg expressed in yeast) HPV (papilloma L1 expressed in yeast)

VIRUS LIKE PARTICLES

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Recombinant Vaccine

The gene coding for HBsAg was discovered in 1970.

The gene has been inserted into a yeast cell.

As the yeast cell grows it produces large amounts of HBsAg.

The HBsAg is extracted and purified then incorporated into the vaccine.

Recombinant DNA modified organismsLive Vectors

Cloning of genetic material from one organism into another.

The non virulent parent organism expresses the antigens of the cloned genetic material.

A vaccine would produce a response against the introduced antigen as well as the original organism.

Recombinant DNA modified organisms

Vaccinia virus expressing papilloma virus antigens on its surface.

DNA vaccines Vs Traditional DNA vaccines Vs Traditional vaccinesvaccines

Uses only the DNA from infectious organisms.

Avoid the risk of using actual infectious organism.

Provide both Humoral & Cell mediated immunity

Refrigeration is not required

Uses weakened or killed form of infectious organism.

Create possible risk of the vaccine being fatal.

Provide primarily Humoral immunity

Usually requires Refrigeration.

DNA vaccines Traditional vaccines

HOW DNA VACCINE IS MADEHOW DNA VACCINE IS MADE

Viral gene

Expression plasmid

Plasmid with foreign gene

Recombinant DNA Technology

Bacterial cell

Transform into bacterial cell

Plasmid DNA

Plasmid DNA get Amplified

Plasmid DNA Purified

Ready to use

METHODS OF DELIVERYMETHODS OF DELIVERY

Syringe delivery:-

Either intramuscularly

or

Intradermally

ContdContd....Gene gun delivery-:

Adsorbed plasmid DNA

into gold particles

Ballastically accelerated

into body with gene gun.

HOW DNA VACCINE WORKSHOW DNA VACCINE WORKS

BY TWO PATHWAYSENDOGENOUS :- Antigenic Protein is presented by

cell in which it is produced

EXOGENOUS :- Antigenic Protein is formed in

one cell but presented by

different cell

HOW DNA VACCINES WORKHOW DNA VACCINES WORK

Muscle Cells Plasmid DNA

+

mRNA

Antigenic Protein

Antigenic Peptides

MHC-I

Plasmid DNA

Nucleus

ENDOGENOUS ENDOGENOUS PATHWAYPATHWAY

Multiply

Memory T cells

T- Helper Cell

EXOGENOUS EXOGENOUS PATHWAYPATHWAY

Antigenic Protein come outside

Phagocytosed

Antigen Presenting Cell

Antigenic Peptides

T- Helper Cell

Cytokines

Activated B-Cell Memory B-Cell

Plasma B-Cell

Memory Antibodies

MHC-II

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Recombinant Vaccine

ADVANTAGESADVANTAGES

Produce both Humoral & cell mediated immunity.

Focused on Antigen of interest.Long term immunity.Refrigeration is not required.Stable for storage.Produced more quickly.In larger quantities.Free from infectious virus particles.

DISADVANTAGESDISADVANTAGES

Limited to protein immunogen only.

Extended immunostimulation leads to chronic inflammation.

Some antigen require processing which sometime does not occur.

Genetic ToxicityGenetic Toxicity

Integration of DNA vaccine into host Genome

Insertional mutagenesis Chromosome instability

Turn ON Oncogenes

Turn OFF Tumor suppressor genes

FUTURE PROSPECTSFUTURE PROSPECTS

Plasmid with multiple genes provide immunity against many diseases in one booster.

DNA vaccines against infectious diseases such as AIDS, Rabies, Malaria can be available.

Why no HCV vaccine

HCV has different genotypes: Since hepatitis C has at least six genotypes, several different vaccines would be needed to protect against each genotype.

HCV mutates very easily: This means that some of its genetic code can change a little bit when it replicates itself. The result is a virus that keeps its genotype, but is different enough to confuse a vaccine.

There is no effective small animal model or cell culture system:This makes vaccine development very challenging because researchers can't see how the virus really works in a natural environment. Really, scientists don't truly understand the whole life-cycle of the hepatitis C virus because infecting liver cells (called hepatocytes) is very difficult.

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