Phase 1B 2015 Phil Watson

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Phase 1B2015Phil Watsonp.f.watson@shef.ac.uk

Vaccines Theory and Practice

Learning goals

–

History of Vaccination

Importance of Vaccination

in Healthcare Strategy

Theory of vaccination

Nature of pathogen and vaccine choice

Passive and Active Immunisation

Adjuvants

Different forms of vaccine agent

Live-attenuated

Inactivated

Subunit

Peptides

DNA vaccines

Recombinant vectors

Qualities of an Ideal Vaccine

Vaccines Theory and Practice

Vaccination is one of the most effective weapons in the medicinal armouryWhy?Because it is successful and cost effective (compared to pharmaceuticals)Since the discovery and development of vaccination a number of formerly major afflictions have been controlled or the frequency hugely reduced

Smallpox

Diptheria

Mumps

Tetanus

Pertussis (whooping cough)

Poliomyelitis

Last natural case – 1977 - It is now eradicated

Vaccines work !

Pre-VaccineAnnual MorbidityPercent DecreaseCurrent LevelsDiptheria21,000100%0Mumps162,00099%982Rubella50,00099%4Smallpox29,000100%0Polio16,000100%0

(typical US population data)

Poliomyelitis

Poliomyelitis

Today there is a pressing need for new vaccines

HIV-1 – approximately 16,000 new infections per day

Substantial economic impact of HIV/AIDS

Now a pandemic

Destruction of economies and human capital

No really effective current

vaccine

Treatment – not cure

Anti-

retrovirals

- AZT

Treatments consume the majority of household budget

High numbers of orphans

Breakdown in family structures

S.A. Reported that approximately 20% teachers are living with HIV

Ebola

Highly contagiousNew strains can emerge at any timeDeath rates from 20-90%Modern transport hubs mean that we arenever more than 5 hours from an outbreakin a large population centerBioterrorism

Origins of Vaccination

VariolationObserved in ancient times that infection with a particular disease renders the individual resistant to infection with the same diseaseAncient Greece Thucydides 429 BCChina 900ADSmallpox – Was a worldwide scourgeFatality – 20%Survivors scarred

Infection with a mild case protected the individual from subsequent serious infection

Scratches on the arm inoculated with pus from a pustule -

variolation

Origins of Vaccination

VariolationEdward Jenner –Based on the observation that milkmaids (who often suffered from cowpox) were resistant to infection by smallpoxInfected people deliberately with pus from cowpox lesionThe result was a resistance to smallpoxTested on a boy James Phipps 8 yo

1749-1823

Edward Jenner

Development of Immunological Theory

The

concept of microorganisms as source of disease

Infection

Robert Koch (1843-1910) – the association of particular diseases with a specific variety of microorganism

Louis Pasteur (1822-1895)The idea of generating weakened pathogens to artificially infect subjects – concepts still used todayMost famously developed a vaccine for rabies

Development of Immunological Theory

Immunological Theory

Infection with an organism lead to the generation of protective substances in the serum

This protection persisted "memory"

Could be transferred to other subjects

By late 19th century - protective substance identified as circulating globulin

Could neutralise and kill bacteria and other pathogens

Antibodies

Modern Concept of Immunisation and Vaccines

Manipulating the immune system to generate a persistent protective response against pathogens Immunisation with a vaccine that can trigger an immune response and safely mimic natural infectionMobilise the appropriate arms of the immune system and generate immunological memory

(Active immunisation

)

Passive vs Active Immunization

Passive Immunisation

Transfer of preformed antibodies to the circulation

Can be natural or artificial

Natural Passive Immunity

This occurs naturally by the transfer of maternal antibodies across the placenta to the developing foetus

Provides protection against:

Diptheria

Tetanus

Streptococcus

Rubella

Mumps

Poliovirus

Passive Immunisation

Indications for use of artificial passive immunity

Individuals with

agammaglobulinaemias

– B cell defects – inborn or acquired

Treated with pooled normal human IgG

Exposure to a disease that could cause complications –

eg

. immune compromised patient exposed to measles or other pathogen

Or - when there is no time for active immunization to give protection

ie

. a pathogen with a short incubation time

Acute danger of infection

Anti-toxins and Anti-veninsPrior to the introduction of vaccines and antibiotics, passive immunization was the major treatment for a range of infectious diseasesUsually horse serum – despite the risks These antisera were frequently used to neutralise toxinsWith some pathogens the main hazard is not the primary infection itself Which can be eliminated by the immune systemRather it is the effects of very potent toxins released by the bacteriaTwo common examples are Tetanus Clostridium tetaniandBotulinum Clostridium botulinum

Passive Immunisation

Natural immunity to these toxins is difficult to achieveGiven the lethal dose of botulinum toxin is approximately 1.5 ng/kg intravenousExposure to sufficient toxin to stimulate the immune system would be lethalDeactivated toxin derivatives (toxiods) can be used as vaccines to produce immunity and this will be covered in the section dealing with Active ImmunisationMost commonly used is tetanus toxoid

Passive Immunisation

Passive Immunisation

A painting by Sir Charles Bell dating from the Napoleonic War period. It shows a patient with tetanus resulting from a gunshot wound.

Uses of Passive Immunization

Routinely used for people infected or exposed to BotulismTetanusDiptheriaHepatitisMeaslesRabies

Also – exposure to venom Snake bite InsectsJellyfish

Anti-toxins

Used

prophylactically

to reduced the chance of establishing infection after exposure

Anti-

venins

SUMMARY

Drawbacks of Passive Immunisation

Does not activate immunological memory

No long term protection

Possibility of reaction to anti-sera

Challenging the subjects immune system to induce a state of immunityThe production of high affinity protective antibodies against the immunogenThe induction of immunological memory

Also known as “vaccination” – derived from the Greek word for cow and based on the historical work of Jenner on smallpox

Not to be confused with “inoculation” - which strictly speaking refers to the introduction of viable microorganisms into the subject.

Active Immunisation

Aims of a “perfect” vaccineTo achieve long term protection (ideally from a small number of immunisations) To stimulate both B and T cellsTo induce memory B and T cellsTo stimulate protective high affinity IgG production (possibly IgA too!)The aim of vaccination depends on the nature of the targeted pathogen and the natural history of the diseaseInduction of Immune ResponseMemorySelection of Appropriate Immune ResponseThe importance of the memory B cell response depends on the nature of the pathogen!

Active Immunisation

Typical Primary and Secondary Antibody Response to Antigen

Initial response –

Relies on “innate” immune systemIgM predominatesLow affinityEssentially germline repertoire

Secondary response Rapid and LargeHigh Affinity IgGSomatic HypermutationT cell helpDoes not rely on innate immune system

The goal of immunisation is to achieve this initial exposure –

without the risks of actual infection!!

Typical Primary and Secondary Antibody Response to Antigen

Second exposure can be years later

During this phase memory T and B cells are generated and circulate for years

Second response is

prompt and powerful

High levels of IgG

High affinity IgG

Somatic hypermutation

T cell help!

Immunological Memory and Pathogens

Influenza – has a rapid onset – Infection can become established before immunological memory can be activated Infection of tissues is blocked by antibodyTherefore it is important to maintain high levels of neutralising antibody by repeated (annual) immunizationCirculating antibodies need to be regularly boostedAlso - annual "escape" variants require the generation of new vaccines

Contrast this with

Polio

– where it takes 3 days to establish infection in the nervous system

This lag provides an opportunity for for memory to be activated and the production of neutralising antibodies

Aim To achieve long term protection (ideally from a small number of inoculations) To induce memory B and T cellsTo stimulate protective IgG production (or IgA)Some pathogens infect primarily through mucous membranes!

Active Immunisation

Active Immunisation

The first stage of any immunisation is to engage the Innate Immune System

Elicit “danger” signals that activate the immune systemtriggers such as molecular fingerprints of infection – PAMPs (pathogen-associated molecular patterns) etc..Engage TLR receptors

Activate specialist antigen presenting cells – eg. follicular dendritic cells

Engage the Adaptive Immune System

Generate memory T and B cells

Activate T cell help

Whole OrganismLive attenuated pathogenKilled, inactivated pathogenSubunitToxoidsAntigenic ExtractsRecombinant proteinsPeptidesDNA VaccinesEngineered Virus

Active Immunisation

Pros and Cons !

Different Vaccine Types

Examples of Common Vaccines

Whole Organism

Bacterial CellsAnthrax InactivatedCholera InactivatedPertussis InactivatedTuberculosis Live Attenuated (BCG)Typhoid Live Attenuated

Viral Particles

Hepatitis A Inactivated

Influenza Inactivated

Measles Live Attenuated

Mumps Live Attenuated

Polio Sabin Live Attenuated

Polio Salk Inactivated

Live Attenuated VaccinesMethods of Attenuating Pathogens

Tuberculosis – BCGBacillus Calmette-Guerin (BCG)Mycobacterium bovis – grown for 13 years on medium containing bileBecame adapted and had reduced virulence

Polio Sabin

Polio virus grown on monkey kidney epithelial cells

Prolonged culture leads to adaptation and a strain that has reduced virulence in humans

Other methods can include chemical treatments

Live Attenuated VaccinesAdvantages

Attenuated pathogen sets up a transient infectionActivation of full natural immune responseProlonged contact with the immune systemThe stimulation of a memory response in the T and B cell compartmentsResulting in prolonged and comprehensive protection

Often only a single immunization is required – advantages in the Third World

Live AttenuatedDisadvantages

Immunocompromised patients (or other rare individuals) may become infected as a result of immunizationComplications !For example – live measles vaccine1 per 1,000,000 - post-infectious encephalomyelitis(0.5 – 1.0 per 1000 with natural disease)

Occasionally the attenuated organism can revert to a virulent form

For example –

Polio Sabin

Approximately 1 case in 2,400,000 doses

In areas with poor sanitation this can lead to a serious outbreak – so Polio Salk is the preferred vaccine in the Third World

Live AttenuatedDisadvantages

Refrigeration and Transport!

Typically live organisms need to be refrigerated for stable storage

This can be an issue in remote areas of the world

This is an important issue for any vaccine

Can it be used without extensive technology?

Is it stable?

Correct Vaccine Preparation is Critical with Live Attenuated Organisms

Salk Polio vaccine is prepared by inactivation of viral particles by formaldehyde treatment1950s - A series of outbreaks were caused by improper chemical treatment

It is important that the treatment does not reduce the immunogenicity of the pathogen – to ensure a correct response

Heat treatment is not preferred as it can alter conformation of target antigens

Modern approaches can exploit recombinant DNA technology to remove genes that control virulence but leave intact the genes for infection

Whole Inactivated Pathogen

No risk of infectionStorage less criticalA wide range of different antigenic components are present so a good immune response is possible

Advantages

Whole Inactivated Pathogen

Disadvantages

Tend to just activate

humoral

responses

Lack of T cell involvement

Without transient infection the immune response can be quite weak

Repeated booster vaccinations required

Patient compliance can be an issue

Examples of Inactivated Vaccines

BacterialAnthraxCholeraPertussisPlagueVirusesHepatitis AInfluenzaPolio (Salk)RabiesRubella

Not as effective as live attenuated in producing cell-mediated immunity

ie

. they are less effective at activating T cell immunity

Typically, repeated doses are necessary to provide protection

Subunit Vaccines

Theoretically safer than handling live or inactivated pathogensNo risk of infectionPurify molecular components as immunogenic agent

Currently 3 major types of such vaccines are in use –

Inactivated

exotoxins

(

toxoids

)

Capsular polysaccharides

Recombinant microbial antigens

Peptide vaccines

Bacterial Exotoxins

A number of important pathogens produce the symptoms of disease as a result of exotoxinsFor example diptheria and tetanus

Diptheria toxin inactivates mammalian elongation factor EF2So it is an inhibitor of translationLethal dose – approx 100 ng/kg - Necrosis of the heart and liver

Tetanus toxin – neurotoxinUncontrolled contraction of voluntary muscles

Toxoid

– heat treated or chemically modified to eliminate toxicity

Capsular PSs are highly polar, hydrophilic cell surface polymers consisting of oligosaccharide repeating units. These molecules are the main antigens involved in the protective immunity to encapsulated bacteria. Capsular PSs interfere with bacterial interactions with phagocytes by blocking opsonization. Opsonization is the coating of the organisms by specific antibodies and complement, which enables host phagocytes to ingest and destroy invading bacteria.

Capsular Polysaccharides

Subunit Vaccines

Purified Proteins

Cultivation of pathogen and subsequent processing to purify single component

(

toxoids

are an example)

Recombinant Proteins (since

1970s

)

Cloning and expression of single gene in recombinant host

Examples include subunit vaccine comprised of Hepatitis B surface proteins (expressed in yeast)

Gardasil

– a recombinant vaccine for human

papilloma

virus

Virus coat proteins expressed in yeast

Spontaneously assemble in virus like particles

DifficultiesIt is now clear that knowledge of HLA presentation of peptides is essentialPeptides can be stimulatory OR suppressive !Most B cell epitopes are conformational

Synthetic Peptides as VaccinesDespite initial promise – the field has advanced slowlyAim – to produce a peptide that includes immunodominant B cell epitopes and can stimulate memory T cell development

B cell

T cell

APC

Subunit Vaccines

AdvantagesSafetyOnly portions of pathogen are usedNo risk of infectionMay be easier to store and preserve

Disadvantages

Immune response is less powerful than to live attenuated vaccines

Repeat vaccinations needed and

adjuvants

Subunits have to chosen that elicit a response in the widest range of subjects (HLA differences?)

Adjuvants

Essentially any substance added to vaccine to stimulate the immune system

Can include:

Whole killed organisms

Toxoids

Proteins (as in conjugate vaccines)

Chemicals

Aluminium salts

Paraffin oil

Adjuvants

The mechanisms can vary:

Aluminium salts may extend the half-life of immunogen in the site of injection (depot effect)

Chemicals can cause irritation and inflammation

Toxoids

and killed organisms trigger the immune system and send out “danger signals”

Aim – to transiently express genes from pathogens in host cells. Generates immune response similar to natural infection Leading to T and B cell memory responses

Expression vector

Transfected

into muscle cells

Expression from

episomal

form or DNA may integrate into chromosome

Other cell types may also take-up DNA – such as antigen presenting cells (follicular dendritic cells)

DNA Vaccines

DNA Vaccines

As proof of the principle of DNA vaccination, immune responses in animals have been obtained using genes from a variety of infectious agents, including Influenza virus,Hepatitis B virusHIV,Rabies virus

In some cases protective responses have resulted

Still too little is known about how DNA immunisation leads to immune response

What

adjuvants

could be useful?

Possible side effects?

Advantages:DNA vaccines do not require complex storage and transportationDelivery can be simple and adaptable to widespread vaccination programs – “DNA gun”

DNA Vaccines

Disadvantages:

As with “killed” vaccines, and subunit vaccines, there is

no transient infection

DNA

vaccination is likely to produce a mild immune response and require subsequent boosting

Recombinant Vector VaccinesAim – to imitate the effects of transient infection with pathogen but using a non-pathogenic organismGenes for major pathogen antigens are introduced into a non-pathogenic or attenuated microorganism and introduced into the hostViral or BacterialVaccinia virusCanarypoxAttenuated poliovirusAttenuated strains of SalmonellaBCG strain of Mycobacterium bovis

Canarypox

ALVAC

-HIV

Recombinant Viral

Vaccines

Adenovirus

Strains that can infect humans

Vesicular Stomatitis Virus

relative of rabies virus

Advantages

Create ideal stimulus to immune system

Produce immunological memory

Flexible - different components can be engineered in

Safe - relative to live attenuated pathogen

Potential drawbacks:

Require refrigeration for transport

Can cause illness in compromised individuals

Immune response to virus in subjects can negate effectiveness

The ideal vaccine

Safe!

this could mean attenuated live if suitable or subunit if the pathogen is lethal for example

Should

induce a suitable immune response

for example - mucosal if pathogen uses this route

high antibody titer if antibody most useful protective agent

Generate T and B cell memory

Stable and easy

to transport

for use in remote areas

Should not require

repeated boosting

patient compliance