Vaccines Theory and Practice Phase 1B 2015 Phil Watson [email protected] 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 armoury Why? 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 Dipther ia Mumps Tetanus Pertussis (whooping cough) Poliomyeli tis Last natural case 1977 Smallp eradicated ox - It is now
Vaccines work ! PreVaccine Annual Morbidity Percent Decrease Current Levels Diptheria 21,000 100%
0 Mumps 162,000 99% 982 Rubella 50,000 99%
4 Smallpox 29,000 100% 0 100% 0 Polio
16,000 (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 contagious New strains can emerge at any time Death rates from 20-90% Modern transport hubs mean that we are never more than 5 hours from an outbreak in a large population center Bioterrorism
Origins of Vaccination Variolation Observed in ancient times that infection with a particular disease renders the individual resistant to infection with the same disease Ancient Greece China 900AD
Thucydides 429 BC Smallpox Was a worldwide scourge Fatality 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 Variolation 1749-1823 Edward Jenner Jenner Based on the observation that milkmaids (who often suffered from cowpox) were resistant to infection by smallpox Edward Infected The people deliberately with pus from cowpox lesion
result was a resistance to smallpox Tested on a boy James Phipps 8 yo 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
Development of Immunological Theory Louis Pasteur (1822-1895) The idea of generating weakened pathogens to artificially infect subjects concepts still used today Most
famously developed a vaccine for rabies 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 (Active immunisation) 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 infection
Mobilise the appropriate arms 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 Passive Immunisation Anti-toxins and Anti-venins Prior to the introduction of vaccines and antibiotics, passive immunization was the major treatment for a
range of infectious diseases Usually horse serum despite the risks These antisera were frequently used to neutralise toxins With some pathogens the main hazard is not the primary infection itself Which can be eliminated by the immune system Rather it is the effects of very potent toxins released by the bacteria Two common examples are Passive Immunisation Natural immunity to these toxins is difficult to achieve Given the lethal dose of botulinum toxin is approximately 1.5 ng/kg intravenous Exposure to sufficient toxin to stimulate the immune system would be lethal
Deactivated toxin derivatives (toxiods) can be used as vaccines to produce immunity and this will be covered in the section dealing with Active Immunisation Most commonly used is tetanus toxoid 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. SUMMARY Uses of Passive Immunization Routinely used for people infected or exposed to
Botulism Tetanus Diptheria Anti-toxins Hepatitis Measles Rabies Used prophylactically to reduced the chance of establishing infection after exposure Also exposure to venom Snake bite
Anti-venins Insects Jellyfish Drawbacks of Passive Immunisation Does not activate immunological memory No long term protection Possibility of reaction to anti-sera Active Immunisation Challenging the subjects immune system to induce a state of immunity
The production of high affinity protective antibodies against the immunogen The 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 vaccine To achieve long term protection (ideally from a small number of immunisations) To stimulate both B and T cells To induce memory B and T cells
To 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 disease Induction of Immune Response Memory Selection of Appropriate Immune Response The importance of the memory B cell response depends on the nature of the pathogen! Typical Primary and Secondary Antibody Response to Antig Initial response Relies on innate immune system IgM predominates Low affinity
Essentially germline repertoire The goal of immunisation is to achieve this initial exposure without the risks of actual infection!! Secondary response Rapid and Large High Affinity IgG Somatic Hypermutation
T cell help Does not rely on innate immune system Typical Primary and Secondary Antibody Response to Antig 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! Second exposure can be years later 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 antibody Therefore it is important to maintain high levels of neutralising antibody by repeated (annual) immunization Circulating antibodies need to be regularly boosted
Also - 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 Active Immunisation Aim To achieve long term protection (ideally from a small number of inoculations) To induce memory B and T cells To stimulate protective IgG production (or IgA) Some pathogens infect primarily through mucous membranes!
Active Immunisation The first stage of any immunisation is to engage the Innate Immune System Elicit danger signals that activate the immune system triggers 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 Active Immunisation
Different Vaccine Types Whole Organism Live attenuated pathogen Killed, inactivated pathogen Subunit Toxoids Antigenic Extracts Recombinant proteins Peptides DNA Vaccines Engineered Virus Pros and Cons ! Examples of Common Vaccines
Whole Organism Bacterial Cells Anthrax Inactivated Cholera Inactivated Pertussis Inactivated Tuberculosis 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 Vaccines Methods of Attenuating Pathogens Tuberculosis BCG Bacillus Calmette-Guerin (BCG) Mycobacterium bovis grown for 13 years on medium containing bile Became 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 Vaccines Advantages Attenuated pathogen sets up a transient infection Activation of full natural immune response Prolonged contact with the immune system The stimulation of a memory response in the T and B cell compartments Resulting in prolonged and comprehensive protection
Often only a single immunization is required advantages in the Third World Live Attenuated Disadvantages Immunocompromised patients (or other rare individuals) may become infected as a result of immunization Complications ! For example live measles vaccine 1 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 Live Attenuated Disadvantages 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 treatment
1950s - 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 Advantages No risk of infection
Storage less critical A wide range of different antigenic components are present so a good immune response is possible 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
Bacterial Anthrax Cholera Pertussis Plague Viruses Hepatitis A Influenza Polio (Salk) Rabies Rubella Not as effective as live attenuated in producing cellmediated 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 pathogens No risk of infection Purify 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 exotoxins For example diptheria and tetanus Diptheria toxin inactivates mammalian elongation factor EF2 So it is an inhibitor of translation Lethal dose approx 100 ng/kg Necrosis of the heart and liver Tetanus toxin neurotoxin
Uncontrolled contraction of voluntary muscles Toxoid heat treated or chemically modified to eliminate toxic Capsular Polysaccharides 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. 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 Synthetic Peptides as Vaccines Despite initial promise the field has advanced slowly Aim to produce a peptide that includes immunodominant B cell epitopes and can stimulate memory T cell development APC B cell T cell Difficulties
It is now clear that knowledge of HLA presentation of peptides is essential Peptides can be stimulatory OR suppressive ! Most B cell epitopes are conformational Subunit Vaccines Advantages Safety Only portions of pathogen are used No risk of infection May 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 halflife 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 DNA Vaccines 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 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 virus HIV, 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?
DNA Vaccines Advantages: DNA vaccines do not require complex storage and transportation Delivery can be simple and adaptable to widespread vaccination programs DNA gun 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 Recombinant Vector Vaccines Aim to imitate the effects of transient infection with pathogen but using a non-pathogenic organism Genes for major pathogen antigens are introduced into a non-pathogenic or attenuated microorganism and introduced into the host Viral or Bacterial Vaccinia virus Canarypox
Attenuated poliovirus Attenuated strains of Salmonella BCG 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
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