A vaccine is any preparation of dead or attenuated pathogens, or their products, that when introduced into the body, stimulates the production of protective antibodies or T-cells without causing the disease

The terms vaccination and vaccine derive from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows; vacca, cow) protected them from the highly contagious and frequently fatal disease smallpox

He tested his theory in 1796 by inoculating 8-year-old James Phipps with liquid from cowpox pustule

Subsequent inoculation of the boy with smallpox produced no disease

Since Jenner's time, the term has been retained for any preparation intended to attain the same

Edward Jenner (1749-1823)

However, this approach dates back, well before Jenner’s time in India and China where they performed what is known as variolation Variola virus=smallpox virus

Variolation, a procedure developed in China and India 1000 AD used a live smallpox vaccine to generate immunity

Employing several different techniques ‘well individuals’ were exposed to variolous material from a human

with a milder form of smallpox—presumably in the expectation that this would cause less severe disease in the recipient—an early form of ‘attenuation

Whole organism Live/attenuated vaccines Killed/inactivated vaccines

Toxoids

Peptide vaccines

Recombinant vaccines

DNA vaccines

Live/attenuated vaccines make up the bulk of successful viral vaccines

Are prepared from attenuated strains that are almost or completely devoid of pathogenicity but are still immunogenic

They multiply in the human host and provide continuous antigenic stimulation over a period of time

Use of a related virus from another animal – e.g. the use of cowpox to prevent smallpox

Administration of pathogenic or partially attenuated virus by an unnatural route

the virulence of the virus is often reduced when administered by an unnatural route

immunization of military recruits against adult respiratory distress syndrome using enterically coated live adenovirus type 4, 7 and (21).

Passage of the virus in an "unnatural host" or host cell – the major vaccines used in man and animals

have all been derived this way After repeated passages, the virus is

administered to the natural host The initial passages are made in healthy

animals or in primary cell cultures Examples: the 17D strain of yellow fever (in

mice and then in chick embryos), Polioviruses (in monkey kidney cells) and measles (in chick embryo fibroblasts)

Development of temperature sensitive mutants this method may be used in conjunction with

the above method

The vaccine is injected sc/im, virions enter various cell types (APCs) using receptor-mediated endocytosis

Proteolytic degradation of viral proteins occurs, the peptides produced are then loaded onto MHC I molecules

The complex is displayed on the cell surface

Circulating cytotoxic T cells recognize the complex, become activated and and release cytokines

The cytokines trigger apoptosis (programmed suicide) of the infected cells

Some Tc become memory cells but the basis of this is incompletely understood

Additionally, immature DCs will phagocytose virus vaccine initiating a series of events that leads to the production of plasma cells, neutralizing IgG antibodies and memory B cells

One important advantage of live/ attenuated vaccines is that they are sufficiently immunogenic therefore primary vaccine failure are uncommon and are usually the result of inadequate storage or administration

Several potential safety problems exist with live/attenuated vaccines:

Underattenuation Mutation leading to reversion to virulence Preparation instability Contaminating viruses in cultured cells Heat lability administration to immunocompromized or

pregnant patients may be dangerous

The term killed generally refers to bacterial vaccines whereas inactivated relates to viral vaccines

An inactivated whole organism vaccine uses pathogens which are killed and are no longer capable of replicating within the host

The pathogens are inactivated by heat or chemical means while assuring that the surface antigens are intact

Inactivated vaccines are generally safe, but are not entirely risk free

Organism Method of inactivation

Rabies β-propiolactone

Influenza β-propiolactone

Polio Formaldehyde

Hepatits A formaldehyde

Organism Method of inactivation

Salmonella typhi Heat plus phenol or acetone

Vibrio cholera Heat

Bordetella pertusis Heat or formaldehyde

E.Coli (experimental) Colicin

Yersinia pestis Formaldehyde

Following injection, the whole organism is phagocytosed by immature dendritic cells

Processed peptides will be presented on the cell surface as separate MHC II:antigenic fragment complexes

Th2, each with a TCR for a separate antigenic fragment will be activated

B cells, each with a BCR for a separate antigenic fragment will bind antigens that drain along lymph channels

The separate antigens will be internalized and presented as an MHC II:antigenic fragment

This will lead to linked recognition with the appropriate Th2

Activated Th2 will release IL2, IL4 IL5 and IL6, inducing B-cell activation, differentiation and proliferation with subsequent isotype switch (IgM to IgG) and memory B cell formation.

First, they are safe because they cannot cause the disease they prevent and there is no possibility of reversion to virulence

Second, because the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals

Third, they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines are used out in the community

Fourth, all the antigens associated with infection are present and will result in antibodies being produced against each of them

Contamination by toxins or chemicals Allergic reactions

Surface endotoxins on inactivated pertussis vaccine occasionally induce DTH responses, and influenza virus has been linked to similar reactions, though this may be due more to the immunogenicity of the egg whites in which the virions are raised

Autoimmunity

Also, inactivated vaccines do not always induce protective immunity. Multiple boosters and an adjuvant are

usually necessary for continual antigen exposure

the dead organism is incapable of sustaining itself in the host, and is quickly cleared by the immune sysytem

Furthermore, inactivated vaccines are generally capable of inducing humoral immunity rather than cellular immunity.

A toxoid is a chemically or physically modified toxin that is no longer harmful but retains immunogenicity

Certain pathogens cause disease by secreting an exotoxin: these include tetanus, diphtheria, botulism and

cholera In addition, some infections, for example

pertussis, appear to be partly toxin mediated Specific physical or chemical modification of

the toxins produces a toxoid, which is a vaccine

The principal toxin is tetanospasmin Binds to specific membrane receptors located

only on presynaptic motor nerve cells Internalization and migration of this toxin to

the CNS blocks the metabolism of glycine which is essential for the normal functioning of gama amino butyric acid (GABA) neurons

GABA neurons are inhibitory for motor neurons

Their non-functioning results in excess activity in motor neurons

This gives rise to muscle spasms, a characteristic feature of tetanus

Manufactured by growing a highly toxigenic strain of Clostridium tetani in a semi-synthetic medium

Bacterial growth and subsequent lysis release the toxin into the supernatant

Formaldehyde treatment converts the toxin to a toxoid by altering particular amino acids and inducing minor molecular conformational changes

Ultrafiltration then removes unnecessary proteins residues

The toxoid is physicochemically similar to the native toxin thus inducing cross-reacting antibodies

But, the changes induced by formaldehyde treatment render it non-toxigenic

Upon administration (sc/im) the toxoid molecules are taken up at the vaccination site by immature dendritic cells

Within this cell, they are processed through the endosomal pathway where they are bound to MHC II molecules

The MHC II:toxoid complex then migrates to the cell surface

mature DCs migrate along lymph channels to the draining lymph node

There , they encounter naïve Th 2 cells Identifying and then binding of the MHC

II:toxoid to the specific Th2 receptor then activates the naive T cell, causing it to proliferate

Some toxoid molecules not taken up by DCs pass along lymph channels to the same draining lymph nodes

There, they come into contact with B cells

Binding to the B cell through the specific immunoglobulin receptor that recognizes the toxoid

The toxoid is internalized, processing through the endosomal pathway and presented on the cell surface as an MHC II:toxoid complex as happens in the DCs

These two processes occur in the same part of the lymph node

The B cell with the MHC II:toxoid complex on its surface now comes into contact with the activated Th2 whose receptors are specific for this complex

This process is called linked recognition

The Th2 activates the B cell to become a plasma cell with the production initially of IgM, and then there is an isotype switch to IgG

In addition, a subset of B cells becomes memory cells

The rationale of toxoid vaccination is production of antibodies with enhanced capacity to bind the toxins

They thus form complexes with the toxins preventing then to interact with toxin receptors on the nerve cells (tetanus)

This is referred to as neutralization of the toxins by antibodies

There are three principal advantages: First, they are safe because they cannot

cause the disease they prevent and there is no possibility of reversion to virulence.

Second, because the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals.

Third, they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines are used out in the community

First, they usually need an adjuvant and require several doses (otherwise less immunogenic)

Second, local reactions at the vaccine site are more common—this may be due to the adjuvant or a type III (Arthus) reaction The reaction generally starts as redness and

induration at the injection site several hours after the vaccination and resolve usually within 48–72 h

The reaction results from excess antibody at the site complexing with toxoid molecules activating complement by the classical pathway causing

an acute local inflammatory reaction

A recombinant vaccine contains either a protein or a gene encoding a protein of a pathogen origin that is immunogenic and critical to the pathogen function

The vaccine is produced using recombinant DNA technology

The vaccines based on recombinant proteins are also called subunit vaccines e.g. RTS,S malaria vaccine, passed phase II

now entering phase III

The logic of such vaccines, in simple terms, is as follows: Proteins are generally immunogenic, and

many of them are critical for the pathogenic organism

The genes encoding such proteins can be identified and isolated from a pathogen and expressed in E. coli or some other suitable host for a mass production of the proteins

The proteins of interest are then purified and mixed with suitable stabilizers and adjuvants, if required, and used for immunization

The first step is to identify a protein that is both immunogenic and critical for the pathogen

The gene encoding this protein is then identified and isolated

The gene is integrated into a suitable expression vector and introduced into a suitable host where it expresses the protein in large quantities

The protein is then isolated and purified from the culture system

It is used for the preparation of vaccine

1. Genetically engineered microorganisms, e.g., yeast for the expression of hepatitis B surface antigen (HBsAg) used as vaccine against hepatitis B virus

2. Cultured animal cells, e.g., HBsAg expressed in CHO (Chinese hamster ovary) cell line and C-127 cell line

3. Transgenic plants, e.g., HBsAg, HIV-l (human immunodeficiency virus-I) epitope (in experimental stages)

4. Insect larvae; the gene is integrated into a bacculovirus genome, which is used to infect insect larvae. Often a very high quantity of the recombinant protein is produced .

An alternative application or recombinant technology is the production of hybrid virus vaccines e.g. HBsAg vaccine

Here, vaccinia virus is used as a carrier for genes that encode antigenic proteins of interest

The genes may be derived from organisms which are difficult to grow or inherently dangerous, and the constructs themselves are replication deficient, nonintegrating, stable and relatively easy to prepare

DNA sequence coding for the foreign gene is inserted into the plasmid vector along with a vaccinia virus promoter and vaccinia thymidine kinase sequences

The resultant recombination vector is then introduced into cells infected with vaccinia virus to generate a virus that expresses the foreign gene

The recombinant virus vaccine can then multiply in infected cells and produce the antigens of a wide range of viruses

The genes of several viruses can be inserted, so the potential exists for producing polyvalent live vaccines

Proteins encoded by these genes are appropriately expressed in vivo with respect to glycosylation and secretion

They are processed for major histocompatibility complex (MHC) presentation by the infected cells, thus effectively endowing the host with both humoral immunity and CMI

Small peptide sequences corresponding to important epitopes on a microbial antigen can be synthesized readily economically

Some long ones are also being invented, but are more expensive to manufacture eg. Pf MSP-3 long synthetic peptide (now in

phase II)

Synthetic peptides can be highly immunogenic in their free form provided they contain, in addition to the B cell epitope, T- cell epitopes recognized by T-helper cells

The T-cell epitope must be linked to the B-cell epitope

Such T-cell epitopes can be provided by carrier protein molecules, foreign antigens or within the synthetic peptide molecule itself

The antigens are precisely defined and free from unnecessary components which may be associated with side effects

They are stable and relatively cheap to manufacture

Furthermore, less quality assurance is required Feasible even if the pathogen cannot be

cultivated

Changes due to natural variation of the virus can be readily accommodated

DNA vaccines are usually circular plasmids (supercoiled) that include a gene encoding the target antigen (or antigens) under the transcriptional control of a promoter region active in human cells

With DNA vaccines, the subject is not injected with the antigen but with DNA encoding the antigen

DNA vaccines are composed of a bacterial plasmids

Expression plasmids used in DNA-based vaccination normally contain two units: Antigen expression unit composed of

promoter/enhancer sequences followed by antigen-encoding and polyadenylation sequences

The production unit composed of of bacterial sequences necessary for plasmid amplification and selection

The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology

Sometimes DNA sequences encoding costimulatory molecules sequences that target the expressed protein

to specific intracellular locations (e.g., endoplasmic reticulum)

Once constructed, the vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies

The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from the much larger bacterial DNA and other bacterial impurities

This purified DNA acts as the vaccine

The DNA vaccine can be injected into a muscle just as conventional vaccines are

Using ordinary syringe or gene gun

DNA vaccines elicit cell-mediated as well as antibody-mediated immune responses

A plasmid vector that expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter is injected into the skin or muscle of the the host

After uptake of the plasmid, the protein is produced endogenously

The protein is processed intracellularly into small antigenic peptides by the host proteases and presented to the cell surface with MHC I

Subsequent CD8+ cytotoxic T cells (CTL) are stimulated and they evoke cell-mediated immunity

CTLs inhibit viruses through both cytolysis of infected cells and noncytolysis mechanisms such as cytokine production

The foreign protein can also be presented by the MHC class II pathway by APCs which elicit helper T cells (CD4+) responses

Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated to produce antibodies production

This is the same manner in which traditional vaccines work

The plasmid is taken up by an antigen-presenting cell (APC) like a dendritic cell

The gene(s) encoding the various components are transcribed and translated

The protein products are degraded into peptides

These are exposed at the cell surface nestled in class I histocompatibility molecules

MHC I-peptide complex serves as a powerful stimulant for the development of cell-mediated immunity

If the plasmid is taken up by other cells (e.g. muscle cells)

The proteins synthesized are released and can be engulfed by antigen-presenting cells (including B cells)

In this case, the proteins are degraded in the class II pathway and presented to helper T cells

These secrete lymphokines that aid B cells to produce antibodies

http://biology.kenyon.edu/slonc/bio38/scuderi/ref.html

So far, most of the work on DNA vaccines has been done in mice where they have proved able to protect them against tuberculosis, SARS, smallpox, and other intracellular pathogens

Different DNA vaccines against HIV-1 — the cause of AIDS — are in clinical trials

Delivery of the DNA to cells is still not optimal, particularly in larger animals

The possibility that exists with all gene

therapy, that the vaccine's DNA will be integrated into host chromosomes and will either turn on oncogenes or turn off tumor suppressor genes

Antibiotic resistance?

Extended immuno-stimulation by the foreign antigen could in theory provoke chronic inflammation or autoantibody production  

Thousands of individual steps (summary) Recognize the disease and Identify the

etiologic agent Attempt to grow the agent in laboratory Establish an animal model for disease Identify an immunologic correlate for

immunity to the disease- Usually a serum antibody

choose antigen (in laboratory) Prepare candidate vaccine following Good

Manufacturing Practice

Evaluate candidate vaccine for ability to protect animal model

Prepare protocol for human studies Phase I human trials- Safety and

immunogenicity using small group (phase Ia in company area)

Phase II trials- Safety and immunogenicity using relatively larger group

Phase III trial- Efficacy and safety Long and complicated process

Usually takes 10–15 years Many vaccine candidates fail for every success

The vaccine:•  Attenuated organisms revert to wild type (e.g. polio types 2,3)• ‘Killed’ organisms not properly killed (has happened with polio)•  Inclusion of toxic material (e.g. typhoid, pertussis)•  Contamination by animal viruses•  Contamination by egg proteins (hypersensitivity)•  Cross-reaction with ‘self’ (autoimmunity)

The patient:•   Immunodeficiency (attenuated organisms may cause serious/fatal disease)•  Local inflammatory reactions, often to the adjuvant• Worsening of disease by increasing immunopathology  (risk of therapy) • Hypersensitivity to vaccine (e.g. tetanus)•  Interference between vaccines given together (not always) • Induction of inappropriate response (e.g. dengue)

•Public aversion to the risks of adverse effects pushes towards sub-unit vaccines rather than live attenuated

•Pathogens change their antigens (or have distinct life cycle stages)a) makes initial composition of the vaccine components complicatedb) make a successful vaccine redundant eg. antigenic drift/shift in influenza, (HIV)

•Success so far has been where naturally occurring immunity is strong eg childhood viruses BUT this is not the case for HIV, TB, malaria etc –so vaccine has to be even better

•Successes to date mostly with antibody inducing vaccines- not cell mediated!

•Confounding effects in tropical populations- eg AIDS, malnutrition, pre-existing exposure to environmental organisms (eg re BCG)- parasitic diseases (Th1 vs Th2 balances)

Does deworming prior to vaccination enhance BCG vaccine?Elias et al., Clin Exp Immunol. 2001 123: 219-25

albendazole

Summary•Early vaccines were mostly whole dead or live attenuated •These provide their own integrated adjuvant/ antigens•Now the shift is towards defined subunit-based vaccines•This requires you to select which adjuvant/ antigen to include•Immunology is now having a greater impact on vaccine design:-optimized activation (e.g. TLR synergy)-temporary removal of immune-regulation? (e.g. anti-IL10)•Safety, efficacy and memory are the key!

Peter Delves, Seamus Martin, Dennis Burton and Ivan Roitt: Roitt’s Essential immunology, 11th edition 2006 page 287-311

David Baxter. Active and passive immunity, vaccine types, excipients and licensing: Occupational Medicine 2007;57:552–556

Medical immunology 2006: edited by Gabriel Virella. ‑‑ 6th ed

Goering RV, Dockrell HM, Zuckerman M, Wakelin D, Roitt IM, Chiodini PL and Mims C; Mims’ Medical Microbiology 4th Edition, Philadelphia Elsevier (2008) page 519-542

http://www.brown.edu/Courses/Bio_160/Projects1999/vaccineoverview/vaccineoverviewbody.html

http://www.microvet.arizona.edu/Courses/MIC419/Tutorials/vaccines.html

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