Vaccine Discovery and Development in Microbiology

8/13/2019 Vaccine Discovery and Development in Microbiology

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Vaccines, Vaccine discovery and

Development in microbiology

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Learning outcomes

Appreciate health threat posed by infectious disease

Understand what is meant by protective immunity

Recognize the different types of vaccines

Revise Immune effector mechanisms, then we cover

the major concepts: How can vaccines be rationallydesigned? How can we evaluate that design? What

are the mechanisms of action/protection? The

importance of the concept of "Correlates of

protection" is highlighted

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The threat to human health of infectious disease

Infectious disease kills 60m per annum prematurely

Disproportionate impact on poorest nations

2.1m people die per annum of vaccine preventable diseases

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The impact of immunization programs

Immunization is probably the most cost-effective

health intervention (life saving, reduces burden on

health systems, allows productive contribution, few

administrations, highly effective) Every $1 spent on immunization saves $20 on health

care in the US

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RevisionImmune response

Remember the two historical observations

1. The Peloponnesian war: ThucydidesDuring the plague of Athens (430BC) people who had

recovered from plague were able to treat sufferers

without falling ill a second time. Why?immunity is

adaptive

2. Faroe Islands: Ludwig Panum

In 1781 and 1846 measles struck these isolated

islanders. Only over 65s who had measles in 1841were unaffected in 1846. Why?Immunological

memory

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Why use vaccines

Vaccination is a highly effective approach to diseasecontrol in human and animal health care.

Chemotherapy has been supported by use of vaccines for

a long time

Vaccination is the best known and most successfulapplication of immunological principles to human health

Role of vaccines is increasing as antimicrobial resistance

to drugs becomes more widespread

The quality of human life and well being of fauna is

closely linked to decreasing the likelihood of infection

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Specific adaptive immunity/acquired immunity

Provides a more specific (antigen specific) but

delayed line of defense Highly effective

Diverse

Response adapts (improves over time) Evokes memory

Discriminates between self/non-self

Adaptive immunity links into the effectormechanisms of innate immunity

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Humoral immunity

What happens when antigen meets antibody?

Antibody initiates the Classical complement pathway Antibody opsonizes antigen or microbe for

phagocytosis

Antibody inactivates (neutralizes) antigen or microbe

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Immunity effectors

Humoral immunity: IgM, IgG 1, 2, 3, 4, IgE

Cell-mediated Immunity (CD4 : Th1, Th2, Th17

etc), CD8

Innate Immunity: Complement, cells, other

soluble factors

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Principles of vaccination

Objective of vaccination is to provide effective immunity byestablishing adequate levels of antibody and a primedpopulation of cells which can rapidly expand on renewedcontact with antigen

So that the first contact with the pathogen clearly shouldavoid the pathogenic effect of that agent but still be sufficient

of a stimulus to the immune system Essentially antigen(s) of a vaccine must induce clonal

expansion in specific T and/or B cells, leaving behind apopulation of memory cells. These enable the next encounterwith the same antigen(s) to induce a secondary response

which is more rapid and effective than the normal primaryresponse

The more antigens of the microbe retained in the vaccine, thebetter, and living organisms tend to be more effective thankilled ones

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How do we design a new vaccine

Approach 1. Trial and error

Approach 2. Rational design1) What is the causative agent of disease? How is it

transmitted? Virus, bacterium, intracellular,

extracellular, mode of transmission, vector, animal

reservoir etcWill we ever eliminate a soil organism like C. tetani?

Can we target the reservoir?

2) Which population do we need to protect? And who

should be immunized?

What % of the population do we need to protect to be

effective in public health terms. Can herd immunity help

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3) What is the mechanism of protection? What is the

correlate of protection?

Mechanism= how vaccine protects (e.g. IgG etc)

Correlate of protection = a measurable patient parameter

that accurately predicts protective efficacy

T cells (CD4 or CD8, Th1/Th2), IgG, what titer, against

what epitopes?

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If antibody is the mechanism of protection

What class? IgG, IgM, IgA, IgE (total)

If IgG what subclass: IgG 1, 2, 3, 4 (opsonising,neutralizing, C ?)

What titer?

In serum, saliva, Mucosal surface?

Against what epitopes?

Ig can be a correlate of protection even if not the actual

mechanism

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If T cell response is the mechanism of

protection?

CD8 or CD4 or both If CD4 which subset: Th1, Th2, Th17

Against what epitopes

At which frequency

Serum Ig is easy to measure so even if T cell is the

mechanism, Ig often used as a surrogate

Immunization primes the immune system with the antigenso that a secondary or anamnestic response awaits

pathogen encounter

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4) Implementation

How persistent / long-lived is the protection? Boosters?

Interference? Pre-existing infection etc

Seasonality? Do we need the vaccine to work in a specific

season? (malaria, flu?)

5. LogisticsCold chain, shelf life, cultural influences

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Our new vaccine

Is the population protected?

Measure the correlate of protection Is the vaccine program working?

How long is the protection? Do we need a booster?

Were the population already protected by natural

exposure?

Is wild type pathogen in circulation?

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Criteria for successful vaccines An economic immunogen is available

An understanding of the epidemiology of disease is essential

for the effective use of vaccines

In particular the mode of transmission must be known and

the prevalence of antibody and attack rates in different

cohorts is useful

Effective programs require an effective delivery system

including cold-chain

For a disease which depends on individual to individual

transmission, it is not necessary to achieve 100%

immunization of target population as the reproductive rate

i.e. the # of further cases produced by an infected individual

may fall to less than one if around 70-80% of the population is

immune

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Inactivated / killed vaccines Considerable care must be exercised to ensure that chemical

inactivation, commonly by use of formaldehyde, is effective in

destroying infectivity but does not reduce the immunogenicpotential of the protein

In many cases the immunity conferred by inactivated vaccines is

inferior to that acquired by infection with the virus/pathogen

Also there is some doubt as to the level of cell-mediated immunitystimulated by inactivated vaccines and a need for repeated

boosting of the immunized individual

These vaccines are costly to produce and administer, require high

antigenic dose, and do not confer life long immunity

Further drawbacks include the need for an adjuvant and the

restriction of administration to systemic routes

Thus have limited use against gut or respiratory tract infections

where high levels of secretory IgA required for protection

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Killed vaccines

Disease RemarksViruses Polio Safe in immunocompromised

Rabies Can be given post exposure, with

passive antiserum

Influenza Strain-specific

Hepatitis A Also attenuated vaccine

bacteria Pertusis Potential to cause brain damage

Typhoid About 70% protection

Cholera Protection dubious; may be

combined with toxin subunit

Plague Short term protection only

Q fever Good protection

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Live and attenuated vaccines Natural live vaccines have rarely been used

There are substantial advantages in the use of attenuatedvaccines

The replication of pathogen in restricted host tissues produces

a much larger dose of stimulating antigen

The immune response takes place largely at the site ofinfection

Incase of budding viruses, infected cells stimulate good levels

of cytotoxic memory T-cells

Also the pathogen can be excreted into the environment andtaken up by other susceptible individuals and thus such

vaccines are preferable in an epidemic situation

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Specific advantages of attenuated vaccines Appear to confer lifelong immunity

Induce IgA response in the gut

Allow spread in the community

Effectiveness approaches 100%

Are easily administered and are relatively cheap

Major drawback in the use of attenuated vaccines is the risk

of reversion, that is, during the course of limited growth

within the immunized animal, the genetic changes associated

with attenuation are reversed or nullified by other mutationssuch that a fully infectious pathogen reappears

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Attenuation to vaccine First successfully done by Calmette and Guerin with a bovine

strain (M. bovis) During 13 years (1908-1921) of culture in vitro changed to the

much less virulent form referred BCG (bacille Calmette-Guerin

with some protective effect against TB

Other successes 17 D strain of yellow fever virus through passage in mice and

chicken embryos (1937)

Similar approach with polio, measles, mumps and rubella

Effectiveness of latter vaccines can be shown by the decline of

these conditions over the last two-three decades

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How attenuation obtained Pioneer attenuated organisms obtained purely through

random series of mutations, induced by unfavorable

conditions of growth, constantly monitored and selected forantigen retention and loss of virulence

Tedious process termed genetic roulette

With sequencing capability, emerged the results were widely

divergent e.g. differences between the three types of live(Sabin) polio vaccine

Type 1 polio vaccine contains 57 mutations, and has almostnever reverted to wide type (i.e. virulence)

But type 2 and 3 vaccines depend for their safety only on twomutations. In these latter, frequent reversions to wild typehave occurred resulting in paralytic poliomyelitis

Attenuation now done by site-directed mutagenesis

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Live attenuated vaccines

Disease Remarksviruses Polio Type 2-3 may revert; also

killed vaccine

Measles 80% effective

Mumps

Rubella Now given to both sexes

Yellow fever Stable since 1937

Varicella-zoster Mainly in leukaemia

Hepatitis A Also killed vaccine

bacteria tuberculosis Stable since 1921; also someprotection against leprosy

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Inactivated toxins and toxoids Are the most successful bacterial vaccines

E.g tetanus and diphtheria are based on inactivated exotoxins The same approach could be used for several other infections

Toxin-based vaccines

organism vaccine remarks

Clostridium

tetani

Formalinized

toxin

Alum adjuvant, boost

every 10 years

C. diphtheriae Formalinized

toxin

Usually given with tetanus

Vibrio cholerae Toxin, B

subunit

Sometimes combined with

whole killed organisms

C. perfringens Formalinized

toxin

Newborn lambs

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Current and future developments

Subcellular fragments and surface antigens These are safe and effective vaccines

It is the surface antigens of most organisms that the immune

system sees first and responds, particularly in the case of Bcells and antibody

For organisms that can be controlled by antibody response,

surface antigens constitute a safe and effective vaccine

E.g capsulated bacteria, whose polysaccharides can beobtained in commercial quantities

E.g hepatitis B virus, which massively overproduces its surface

coat

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Synthetic peptides Where it can be shown that a small peptide is protective, it may be

convenient to make it synthetically or by cloning its gene into a suitable

expression vector This approach has been highly successful with the HBs antigen, cloned into

yeast cells and now replacing the first generation vaccine which was

laboriously purified from the blood of carriers

The choice of sequence along the native molecule is based on the use of

predictive algorithms for predicting those parts of the molecule likely toproject from the surface of the protein into an aqueous environment

Approach works because:

There are relatively few antigenic sites along the native molecule seen by

the immune system

Short peptides in solution have a preferred conformation, i.e. there are

sufficient molecules in a conformation that can be recognized by the

immune system and react with antibodies

This approach allows manipulation of the immune response not easily

obtained by immunization with larger protein structures i.e. antibody can

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Synthetic peptidesContd Attractive feature of this approach is that further sequences

can be added e.g. selected B- and T-cell epitopes can be

combined in various ways to optimize the resulting immuneresponse

It is necessary to devise a suitable carrier for immunization

purposes

In animals the most successful experimental example of asynthetic peptide vaccine is against foot and mouth disease:

A short peptide less than 20 amino acids long which acts as an

analogue of a loop region of the viral capsid protein that

stimulates a protective immune response in guinea pigs andcattle

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Recombinant vaccines A further development of the use of gene cloning is to put the

desired gene into some vector which can then be injected into

the patient and allowed to replicate, express the gene and

deliver large amounts of the antigen in situ

Recombinant gene technology now enables the expression of

whole or part of a viral/bacterial/pathogen protein in a variety

of expression systems. The most useful are :

Yeast cells

Transfected eukaryote cells

Insect cells

Cells infected with poxviruses e.g. vaccinia

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Recombinant vaccines The use of vaccinia as a vector is doubtful in humans but

shows considerable promise for delivery of immunogens in aveterinary context, particularly against virus diseases

Concerns include its single use for immunization purposes,

the interference in recipients from pre-existing vaccinia

antibody (many people are already immune to it and would

eliminate it too rapidly) `and the risk of generalized vaccinia

reactions

An alternative viral vector is canarypox

Almost all available attenuated viral vaccines have been

suggested as alternatives

Further suggestion is use of attenuated bacteria as vectors

e.g. BCG

Example of vaccine in clinical trials is the rabies vaccine for

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Anti-Idiotype vaccines Idea is to use mAb technology to make large amounts of anti-

idiotype (anti-Id) against the V region (idiotype) of an Ab of a

proven protective value

The anti-Id, would then have a 3-dimensional shape similar to

the original immunizing antigen and could be used in place of

it

Could be of real value where the original antigen is not itselfsuitable i.e. not immunogenic e.g.

Polysaccharides

Lipid A of bacterial endotoxin (LPS)

Advantage of mAb would be that since it is a protein it should

induce memory, which polysaccharides and lipids normally do

not

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Anti-idiotype vaccines - contd A fraction of the anti-idiotype response contains within its

combining site an internal image of the immunizing antigen

on the surface of the native protein

Such anti-idiotype antibodies have been demonstrated to

protect animals against subsequent challenge with live virus,

e.g. protection of chimps against human diseases such as

hepatitis B and HIV, and experimental infections of mice withreovirus

The reovirus system has been particularly well characterized

and sequencing of the internal image employed to determine

the amino acid sequence of discontinuous viral epitopes

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DNA vaccines Is an alternative approach to a single dose vaccine

DNA itself is injected, coupled to a promoter, into the musclesor skin of the individual to be vaccinated

A plasmid is used a vector

The gene is then expressed in a native conformational state

Excellent immunity, both humoral and cell-mediated, and noevidence for the tolerance that might have been expected to

result from the potentially unlimited source of foreign antigen

There is a lot of interest and activity in this new field and an

influenza vaccine is expected to be tested shortly

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Other developments Include various means of presenting proteins to the immune

system One of this, the so called iscom technology, allows the

generation of more sustained and higher antibody responses

compared to the same vaccines delivered e.g. complexed to

alum adjuvant

The immune-stimulating complexes are particularly successful

when there are problems in delivering proteins which are

normally membrane-bound in virus particles, the latter being

difficult to produce in sufficient amounts for more

conventional approaches

E.g recently introduced vaccine against equine influenza

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Other examples of new vaccines in development include the

use of microsphere technology and immunogens with a

higher immunogenicity

New approaches to vaccine design have concentrated on thedelivery of viral components using novel adjuvant systems

that enhance the B-cell (T-cell) responses to the immunogen

Despite the potential of synthetic peptide vaccines as

immunogens which can selectively stimulate protectiveresponses, the lack of suitable delivery systems has hampered

their development

Recently, the use of polylactide-polyglycoside microspheres

has been shown as effective for the delivery of small peptidesby either the oral or i/m routes

This has inherent flexibility for design of delayed release inocula whereby

pulses of peptides are delivered to immune system on a defined time

schedule after injection, thus enabling development of single dose

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What is Yellow Fever?

An infectious disease that leads to damage of many organs inthe body, often due to severe bleeding

The liver is often affected > jaundice, hence the name Yellow

Fever

Aedes aegypti was identified as key vector There was destruction of mosquito habitats

But was realized that the natural reservoir was monkeys

between which the infection was spread by different jungle

dwelling mosquitoes Occasionally disease was transmitted to humans by different

vectors (jungle/sylvatic yellow feversporadic cases)

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Yellow fever

If the spurious cases from the jungle contacted larger humanpopulations in urban areas, severe epidemics could develop in

which virus was then transmitted byAedes aegyptifrom man

to man

With the development of effective vaccine by Theiler (1937),

the urban form of the disease was eliminated, but epidemics

of jungle form still occur

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Yellow Fever Vaccine discovery

1902 yellow fever causative agent found to be ultra filtrable Virus isolated later

Theiler propagated the virus in brain of mice

He found that repeated passages in mice lead to a progressive

shortening of the incubation time and importantly, asuccessful reduction of the pathogenicity of the virus in

monkeys

Theiler developed a test for measuring protective Abs in mice

and presence of Abs in humans That enabled him to map epidemiology of infections and

evaluate candidate vaccines

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YF Vaccine discovery

He started growing the mouse-adapted virus in chickenembryo cultures

He and others showed that attenuation of virus obtained by

passages in mice was not sufficient

Virus showed diminished viscerotropic properties (mainsource of symptoms of YF) but virus capacity to attack the

brain increased (encephalitis)

Different virus strains were passaged in tissue cultures and

repeatedly tested for their neurotropic activity

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YF Vaccine discovery

A variant of Asibi strain emerged after passage in mincedchicken embryos without CNS (89th114thpassage) that

lacked both viscerotropic and neurotropic effects

Variant was stable and neuro-virulence was not regained

upon repeated passages in chicken embryo cultures First field trials started in 1938 in Brazil

For over 70 years the 17D virus vaccine has proven to be safe

and effective

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YF Vaccine today Vaccine is still produced using original methods-it is passaged

in embryonated chicken eggs and stored as a frozenhomogenate

1951 Max Theiler was awarded the Nobel Prize for YFV

vaccine discovery

in the field of observation, chance only favors the preparedmind (Louis Pasteurs famous dictum)

Why a discovery? Passage of Asibi strain in chicken embryos without CNS

suddenly changed its nature and lost both its viscerotropic

and neurotropic properties

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Development of subunit vaccines - empirical

approach

Subunit vaccines contain one or more pure or semi-pureantigens

In order to develop subunit vaccines, it is critical to identify

those proteins which are important for inducing protection

and to eliminate others The empirical approach to subunit vaccine development,

which includes several steps, begins with pathogen

cultivation, followed by purification into components, and

then testing of antigens for protection

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Subunit vaccine devempirical approach

Empirical approach is time- and labour-consuming, and hasseveral limitations that can lead to failure

Some organisms cannot be cultured

Only allows for the identification of those antigens which can

be obtained in sufficient quantities In some cases, the most abundant proteins are not immuno-

protective.

In other cases, the antigen expressed during in vivo infection

is not expressed during in vitro cultivation

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V i dj


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Vaccine adjuvants

Vaccines containing inert immunogens, e.g. inactivated viral

vaccines, tetanus toxoid require the presence of an adjuvant Adjuvanticity is the adsorption onto, or conjugation to, or

incorporation of antigens into a variety of inert carriers, such asaluminum salts (alum), bentonite, latex or acrylic particles, orlipid lamellae structures (liposomes)

Adjuvants localize the antigen at the site of injection, which is mostoften intramuscular or subcutaneous, and lead to enhancement ofthe immune response by facilitating uptake into macrophages andantigen presenting cells

Saponins are also used, with the effect that a mild inflammatory

reaction at the site of inoculation encourages antigen uptake

The diversity of adjuvants used in veterinary products differs fromthe sole use of aluminium salts in human vaccines

Vaccine Discovery and Development in Microbiology

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