Vaccines, Vaccine discovery and Development in microbiology

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 rationally designed? How can we evaluate that design? What are the mechanisms of action/protection? The importance of the concept of "Correlates of protection" is highlighted

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

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

Revision Immune response

Remember the two historical observations 1. The Peloponnesian war: Thucydides During 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 1841 were unaffected in 1846. Why? Immunological memory
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Why use vaccines

Vaccination is a highly effective approach to disease control 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 successful application 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 is the basis of immunization

1796: Edward Jenner immunizes James Phipps with cowpox then challenges with small pox (1850s Compulsory vaccination in London; 1980: Eradicated) 1885 Pasteur & Germ theory of disease. Rabies vaccine Impact: small pox was a major cause of morbidity and mortality no cases since 1980.

 In 1890, Baron Emil von Behring & Shibasaburo Kitasato discovered substances in blood serum which protected against Diphtheria. Coined the term antibody In 1894, Paul Ehrlich proposed that antibody was; Complementary in shape to the antigen Formed before exposure to antigen Secreted into serum on exposure In 1892, Ilya Metchnikoff discovered phagocytosis, the basis of cell mediated innate immunity In 1900-1906, Sir Almroth Wright, combined both phenomena. Developed vaccine against typhus 1940/50s role of lymphocytes

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 effector mechanisms of innate immunity

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 by establishing adequate levels of antibody and a primed population of cells which can rapidly expand on renewed contact with antigen So that the first contact with the pathogen clearly should avoid 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 a population of memory cells. These enable the next encounter with the same antigen(s) to induce a secondary response which is more rapid and effective than the normal primary response The more antigens of the microbe retained in the vaccine, the better, and living organisms tend to be more effective than killed ones
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By Definition
A vaccine is a molecular or supramolecular agent which elicits specific, protective immunity; i.e. an enhanced adaptive immune response to re-infection by pathogenic microbes through the potentiation of immune memory. The immune system recognizes vaccine agents as foreign, destroys them, and subsequently 'remembers' them. When the pathogenic microorganism is encountered again, the immune system has been primed to respond, by neutralizing the target before it can enter cells, and/or by destroying infected cells before the microorganism can grow and cause damage.
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How do we design a new vaccine

Approach 1. Trial and error Approach 2. Rational design 1) What is the causative agent of disease? How is it transmitted? Virus, bacterium, intracellular, extracellular, mode of transmission, vector, animal reservoir etc Will 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 us
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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 antigen so 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. Logistics Cold 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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Vaccine immunogens/antigens
Inactivated whole pathogens Live attenuated pathogens Microbial product chemically treated to abrogate its pathogenic effect e.g. tetanus toxoid Separated subunits consisting of those microbial proteins responsible for the induction of a protective immune response: under this heading would be included selected gene expression by recombinant gene technology live vectors prepared by recombinant DNA technology and expressing a selected microbial protein known to induce protective immunity e.g. vaccinia virus containing rabies glycoprotein

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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 immunogenic potential 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 immunity stimulated 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 Viruses Polio Rabies Influenza Hepatitis A bacteria Pertusis Typhoid Cholera Plague Q fever Remarks Safe in immunocompromised Can be given post exposure, with passive antiserum Strain-specific Also attenuated vaccine Potential to cause brain damage About 70% protection Protection dubious; may be combined with toxin subunit Short term protection only 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 attenuated vaccines 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 of infection Incase of budding viruses, infected cells stimulate good levels of cytotoxic memory T-cells Also the pathogen can be excreted into the environment and taken 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 mutations such 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 for antigen 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 almost never reverted to wide type (i.e. virulence) But type 2 and 3 vaccines depend for their safety only on two mutations. In these latter, frequent reversions to wild type have occurred resulting in paralytic poliomyelitis Attenuation now done by site-directed mutagenesis
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Live attenuated vaccines

Disease
viruses Polio Measles

Remarks
Type 2-3 may revert; also killed vaccine 80% effective Now given to both sexes Stable since 1937 Mainly in leukaemia Also killed vaccine Stable since 1921; also some protection against leprosy

Mumps
Rubella Yellow fever Varicella-zoster Hepatitis A bacteria tuberculosis

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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 Clostridium tetani C. diphtheriae vaccine Formalinized toxin Formalinized toxin remarks Alum adjuvant, boost every 10 years Usually given with tetanus

Vibrio cholerae
C. perfringens

Toxin, B subunit
Formalinized toxin

Sometimes combined with whole killed organisms

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 B cells 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 be obtained 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 to project 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 31 be made even to otherwise immunosilent regions on native protein

 Synthetic peptides Contd

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 immune response It is necessary to devise a suitable carrier for immunization purposes In animals the most successful experimental example of a synthetic 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 and cattle

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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 a veterinary 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 34 immunization of badgers and foxes in Europe

 Anti-Idiotype vaccines
Idea is to use mAb technology to make large amounts of antiidiotype (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 itself suitable 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 with reovirus 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 muscles or 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 no evidence 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 the delivery 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 protective responses, 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 peptides by 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 vaccines with attendant savings in logistics and cost 39

EXAMPLES: Pass to discovery of Yellow Fever Virus vaccine (1937)

Max Theiler Yellow fever has caused life threatening epidemics through the last 500 years Etiology was established YF virus Max Theiler developed the 17D strain of attenuated virus To the benefit of mankind The concept of discovery

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What is Yellow Fever?

An infectious disease that leads to damage of many organs in the 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 fever sporadic cases)
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 Yellow fever
If the spurious cases from the jungle contacted larger human populations in urban areas, severe epidemics could develop in which virus was then transmitted by Aedes aegypti from 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, a successful 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 chicken embryo cultures He and others showed that attenuation of virus obtained by passages in mice was not sufficient Virus showed diminished viscerotropic properties (main source 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 minced chicken embryos without CNS (89th 114th passage) 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 frozen homogenate 1951 Max Theiler was awarded the Nobel Prize for YFV vaccine discovery in the field of observation, chance only favors the prepared mind (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-pure antigens 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 dev empirical approach

Empirical approach is time- and labour-consuming, and has several 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 immunoprotective. In other cases, the antigen expressed during in vivo infection is not expressed during in vitro cultivation

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Subunit vaccine dev genomics approach (Prediction of protective antigens)

Genomics has revolutionized vaccine research The ability to sequence the whole genome of a virulent microorganism has led some to screen for the most probable protective antigens before confirmatory experiments. This approach is known as reverse vaccinology, and was first used to identify antigens as potential candidate vaccines against serogroup B meningococcus Apart from obvious advantages such as speed and low cost the success of this approach is dependent on the accuracy of antigen prediction, and many bioinformatics tools are available to facilitate this process They can identify surface-associated or outer membrane proteins, signal peptides, lipoproteins, or host-cell binding 50 domains.

Vaccine adjuvants / Adjuvanticity

Adjuvants are materials mixed with vaccines to potentiate the immune response, both humoral &/or cellular, so that a lesser quantity of antigen is required and fewer doses need be given Mechanism of action Prolonged retention and slow release of antigen Activation of macrophages, leading to secretion of lymphokines and attraction of lymphocytes Mitogenicity for lymphocytes
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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 as aluminum salts (alum), bentonite, latex or acrylic particles, or lipid lamellae structures (liposomes) Adjuvants localize the antigen at the site of injection, which is most often intramuscular or subcutaneous, and lead to enhancement of the immune response by facilitating uptake into macrophages and antigen 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 from the sole use of aluminium salts in human vaccines
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