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Vaccine Development Traditional and Modern Approaches Rodney Carbis Head Vaccine Development International vaccine institute 1 Purpose of Vaccination 2 Purpose of Vaccination Protect the individual from disease. Reduce the severity of disease. Protect the community. Eradication of the disease. Reduce the burden of disease. 3 The value of Vaccines Vaccines are the most cost-effective tools for preventing death and disability from infectious disease Luis Fermín Tenorio, the last polio case in the Americas Peru, 1991 Ali Maouw Maalin, last case of smallpox (Somalia,1977) 4 Vaccine Efficacy Diphtheria >87% (industrialized countries) Tetanus Pertussis Oral Polio Measles Hepatitis B Hib 3 doses >95%, 2 doses >80% 80% (wide variation) >90% (lower in tropical countries) >90% (after 12 mths) 85% (at 9 mths) 75 – 95% 90% 5 Financial Benefits of Measles Vaccination Cases averted Lives saved Cases of mental retardation averted Physicians visits saved 52,107,000 5,210 17,360 26,776,000 Hospital days saved 2,972,000 Net benefit achieved 1963 – 1982 USD 5,120,729,000 6 Normal Bacterial Flora Staphylococcus epidermidis Normal colon Bacterial colonization of the body per gm or cm2 Bifidobacterium bifidum Bacterial colonization E.coli 7 Role of Bacterial Toxins in disease DISEASE (BACTERIA) Diphtheria Corynebacterium diphtheriae Tetanus Clostridium tetani Whooping cough Bordetella pertussis Cholera Vibrio cholerae TOXIN PRODUCED Diphtheria toxin Tetanus toxin Adenylate cyclase toxin Pertussis toxin Cholera enterotoxin 8 Invasion of Cells Shigella bacterium invading HELA cell 9 Basic concept of vaccines Deliver to the body some part or all of the disease organism that IMITATES the pathogen but is not pathogenic. – Induce protective immune response. Polysaccharide LPS capsular Entire organism • live (attenuated) • killed Intracellular proteins Surface proteins Toxins 10 New developments in vaccines Identifying the protective antigen – Common antigens – Poorly expressed antigens Expression of antigen – Production system Production of protective antigens in yeast etc. – Vector Live attenuated bacteria Live attenuated viruses 11 New developments in vaccines Antigen presentation Virus like particles Adjuvants Slow release vehicles Conjugation Formulation Multivalent vaccines Addition of muco-adhesives for oral delivery Addition of preservatives and stabilizers (lyophilization) 12 Vaccine manufacture Antigen Production Eggs – Influenza Bacterial / Yeast fermentation – – – Whole organism (e.g. Cholera) Subunit vaccines (e.g. Capsular polysaccharide, Tetanus and Diphtheria toxoid) Genetically engineered proteins (e.g. Hepatitis B and HPV vaccines) Cell culture – – Viral vaccines either whole virus or subunit Genetically engineered proteins 13 Vaccine Processing Antigen concentration. Removal of unwanted foreign components. – – – Host proteins Host DNA Adventitious agents – – – LPS Unwanted proteins (not involved in immunity) Unwanted nucleic acid – Adjuvants, stabilizers, preservatives etc Removal of unnecessary Bacterial or Viral components. Change of diluting solution. Addition of other components. 14 Growth of Virus, Yeast or Bacteria Eggs Clarification Depth filter Concentration + Purification Crossflow filtration Tools available to develop a process Chromatography Ion exchange Bacterial / Yeast Fermentation Cell culture Crossflow filtration Hydrophobic Affinity Ultracentrifugation Centrifuge Size exclusion Chemical Formaldehyde precipitation Inactivation bPL 15 Antigen presentation DT Formulation CONHNHCO(CH2)4CONHNHOC- Vi-CPS CONJUGATES Fill and Finish Combining antigen(s) Combining with adjuvant Stabilizers VIRUS LIKE PARTICLES ISCOMS Preservatives Cryo-protectants ADJUVANTS VIROSOMES 16 Types of Vaccines 17 Types of Vaccines Inactivated toxins Inactivated whole bacteria or viruses Live attenuated bacteria or viruses Subunit vaccines Genetically engineered proteins Polysaccharide vaccines Conjugated vaccines Recombinant DNA modified organisms DNA vaccines 18 Inactivated Toxins Exotoxin – Gram negative and gram positive bacteria. – Heat Labile. – Protein. – Secreted by the bacteria. Endotoxin – – – – Gram negative bacteria. Heat stable. Lipopolysaccharide. Firmly bound to the bacteria outer membrane. 19 Toxoid Not a vaccine against the organism. Vaccine against pathogenic exotoxin. Tetanus, diphtheria, (pertussis?), anthrax? – Purify toxin then chemically inactivate (toxoid) Risk of incomplete inactivation. TT, DT. – Genetically modify toxin so non-toxic CRM (diphtheria), mLT (cholera, ETEC) 20 Inactivated Toxins Tetanus toxin blocks neurotransmission from nerve endings inducing muscle spasm and paralysis Diphtheria toxin inhibits protein synthesis resulting in cell death 21 Tetanus 22 Tetanus Clostridium tetani Gram positive drum stick shaped rod Not transmitted from person to person Transmission via contaminated wounds Found in soil and animal feces Replicate in low oxygen environment Release toxin which enters bloodstream Acts on the nervous system to block neurotransmitters 23 Tetanus The toxin causes the disease. The vaccine consists of toxin which is rendered inactive (toxoid) but retains immunogenicity. The immune response to the vaccine is directed against the toxin rather than the bacteria. Vaccination results in serum antibody production. The antibody neutralizes toxin. Three doses of vaccine recommended to raise antibody levels to protective levels. Immunity lasts about 10 years. Vaccination prevents disease but is incapable of disease eradication. 24 Neonatal and Maternal Tetanus Tetanus in Children has been largely controlled by vaccination. In 1999, WHO estimated 290,000 cases of neonatal tetanus which resulted in 14% (215,000) of neonatal deaths. 30,000 (5%) of Maternal deaths were due to tetanus. Target to eliminate neonatal and maternal deaths due to tetanus by 2005 by vaccination of women of child bearing age. 25 Diphtheria 26 Diphtheria Corynebacterium diphtheriae. Gram positive bacillus. Only produces a powerful exotoxin when infected with a bacteriophage. (the phage carries the toxin gene which coverts the bacteria from non-toxigenic to toxigenic) Diphtheria is now well controlled by national immunization programs. Only receives attention when routine immunization programs fall. 27 Diphtheria 28 Whole Bacteria or Virus Vaccines Cholera bacteria Polio virus 29 Whole Killed Whole pathogen grown and killed – Heat, chemical modification (formaldehyde, bPL,..) – Pertussis, cholera. – IPV (Inactivated Polio), Influenza, Hepatitis A. Advantage – Relatively easy – Generally safe to administer - no risk of reversion, infection. 30 Whole Killed Disadvantages – – – – – – – – – Numerous injections normally required No or limited cellular immunity Immunity often shorter-lasting than live vaccines Reactogenicity from LPS, lipids.. Adjuvant often required (alum, emulsions) Risk of growing pathogenic organism (eg. polio) Risk of incomplete inactivation Inappropriate immune response ? eg RSV ??? Can not focus immunity on protective antigen. 31 Inactivated Whole Cell Cholera Vaccine Manufacturing Process Fermentation SBL Vaccine Cholera toxin Inactivation Concentration And Washing B subunit Bulk Monovalent Formulation 32 Subunit Purify a protein or proteins from pathogen – Eliminate reactogenic contaminants (eg LPS) – Selective presentation of 'protective' antigens Pertussis – Pertussis toxin + filamentous haemagglutinin + pertactin (no LPS) Influenza (subunit) – Mainly haemaglutinin + neuraminidase Disadvantages – Requires growing the pathogen and purifying protective (antigens) subunits. 33 Pertussis 34 Pertussis Bordetella pertussis. Gram negative coccobacillus. Extremely contagious infecting almost all susceptible close contacts. WHO estimated in 1999 that pertussis was still responsible for about 300,000 deaths mainly in sub-Saharan Africa. 40 million cases annually. 35 Pertussis Vaccines Whole cell vaccine. – Whole bacterial cells which are rendered non toxic using formalin. – Problem with this vaccine was its reactogenicity. Acellular pertussis vaccines. – Contain combinations of purified antigens. – In the USA, acellular vaccine was introduced for booster dosing, but in Jan 2000 it was recommended for all doses. 36 Bordetella pertussis Pertussis toxin Pertactin Filamentous haemagglutinin 37 Pertussis components and their role in protection Pertussis toxin: exotoxin secreted during bacterial growth. Binds to receptors on respiratory tract cells. PT is a major virulence factor and is toxic to mammalian cells. Antibody to PT protects mice to lethal challenge. Antibodies to PT are elicited after both vaccination and natural infection. PT is a toxin so must be toxoided for use in a vaccine. 38 Pertussis components Filamentous Haemagglutinin: a cell wall component implicated in attachment to the respiratory epithelium. Antibody to FHA protect mice from lethal respiratory but not intracerebral challenge. Antibody to FHA is produced as a result of infection or after vaccination. Some studies have shown a correlation of antibody titre to FHA with protection whereas other studies have not. 39 Pertussis components Pertacin: a 69 kD outer membrane protein (OMP). Protects neonatal mice against respiratory challenge. Antibody to Pertactin is produced as a result of infection or after vaccination with either whole cell or acellular vaccines. Antibodies against Pertactin are implicated in protection against pertussis. 40 Pertussis Other components being investigated for their role in toxicity and infection. – adenylate cyclase – endotoxin – tracheal cytotoxin – heat labile toxin – agglutinogens 41 Polysaccharide Many bacteria produce a strain-specific capsular polysaccharide on their surface. Antibody to these antigens are protective. – Streptococcus pneumoniae, Haemophilus Type B, Meningitis A,C,W,Y (not B!) Typhoid (Vi). Can be easily purified. Immunogenic in older children / adults. But poorly immunogenic in infants T-cell independent responses Short lived Low antibody responses 42 Different forms of Polysaccharide LPS Capsular polysaccharide 43 Conjugate Vaccines 44 Polysaccharide conjugates 45 Haemophilus b 46 Haemophilus 47 Haemophilus Gram negative rods. Capsulated or non-encapsulated Capsules are polysaccharide. Six distinct serological types (a–f) of encapsulated H. influenzae. 95% of all serious infections attributed to type b (poly ribosyl-ribitol phosphate PRP) Most Hib colonization of the upper respiratory tract are asymptomatic. Disease arises when the bacteria moves into the blood. 48 Haemophilus b Disease Burden (WHO estimates) – >500,000 children die from Hib pneumonia per year. – >500,000 children die from Hib meningitis per year. – 90% of cases are in children under 5 with peak incidence 6 – 11 months. 49 Hib Vaccines Unconjugated vaccines: First Hib vaccines consisted of purified PRP. Immunogenic in children >2 years Immune response in younger children was poor. The response to carbohydrate antigens is T-cell independent. Young children lack the immune system maturity required to mount such a response. 50 Hib Vaccines Conjugated vaccines: Covalently linking PRP to a protein carrier results in T-cell recognition of PRP. Recruitment of T-helper cells allows production of antibodies to PRP and importantly induces immunological memory. This type of response is fully developed at a young age. 51 Typhoid Vaccine Vi Capsular Polysaccharide O COOH O Fermentation AcO AcN O Inactivation Vi Capsular Polysaccharide Purification Sterile Filtration Formulation 52 Vi Conjugate vaccine Vaccine against Typhoid Fever Exoprotein A (rEPA) C=O NHNHCO(CH2)4CONHNH Spacer C=O Vi Capsular Polysaccharide 53 Live Attenuated Most common type of vaccine. OPV (oral polio), measles, mumps, rubella, yellow-fever, rotavirus, influenza, smallpox, BCG, typhoid, anthrax, Live pathogen selected or genetically modified causes no disease or only mild disease. Derived from non-human source (eg cows, monkeys) Selected mild strain from humans Passaged (tissue adapted) strain from humans Genetically modified (limited ability to replicate) 54 Live Attenuated Advantages: – – – – Mimics natural infection Humoral and cellular response. Immunological memory. Generally cheap. Disadvantages: – – – – – Not suitable for all organisms May revert to a virulent form Circulating antibody may interfere with response Immunity to non-protective antigens Safety in immuno-suppressed individuals? 55 Live Attenuated Vaccines Typhoid bacteria Measles virus 56 Measles 57 Burden of Measles Measles remains one of the major childhood killers. WHO estimates 30 million cases of measles per annum. 875,000 children die from measles annually. 58 Measles Caused by Rubeola virus – Genus Morbillivirus – Family Paramyxoviridae Six structural proteins – 3 complexed to the viral RNA – Matrix protein associated with inner layer of lipid membrane – 2 viral membrane proteins Fusion protein – fusion with host cell H protein – adhesion of virus to host cell 59 Immune response to Measles 60 Immune response to Measles Antibody response. – Serum IgM – Serum IgG – Serum IgA and secretory IgAs. Re-exposure induces a strong secondary IgG response and prevents clinical disease. 61 Immune response to Measles Cell Mediated Response – Sensitization of T – lymphocytes. – Production of cytotoxic T- cells. Cellular response is important in recovery from infection. People deficient in this response are at increased risk of death. 62 Measles Vaccine Killed Inactivated vaccine was abandoned in 1967 as it did not provide complete protection. An attenuated vaccine was also being developed and first licensed in 1963. This was found to be too reactogenic for routine use. Further attenuation produced an acceptable vaccine. 63 Attenuation of Measles EDMONSTON B STRAIN 24 passages in primary kidney cells 28 passages in human amnion cells Passages in chick embryo cells Vaccine immunogenic but too reactive. SCHWARZ 85 additional passages in chick embryo cells Vaccine 90-95% effective, low reactogenicity. At least 20 years immunity. 64 Polio Virus 65 Polio Virus An enterovirus belonging to the family picornavirus. Contains RNA surrounded by icosahedral protein capsid. Contains no lipid and is acid stable so can pass through the stomach. Three antigenically distinct types, 1, 2, and 3. Vaccines contain all three types. 66 Oral Vaccination 67 Response to Polio Vaccination 68 OPV vs IPV Oral Polio Vaccine (OPV) Advantages – Results in production of secretory IgA. Important in protecting the individual. Reduces the spread of wild polio. – IgAs has been observed to persist for 5 – 6 years Disadvantage – Genetic instability of Type 3 strain rarely results vaccine induced poliomyelitis. – Transmission of vaccine strains to unvaccinated individuals. 69 OPV vs IPV Inactivated Polio Vaccine – Safe and effective. – Lack of production of mucosal IgAs results in failure to eliminate intestinal re-infection and fecal excretion. – Recommended for countries where polio is no longer endemic. 70 Recombinant Protein produced by genetic engineering Express protective antigen in safe easy-togrow organism – Hepatitis B (HBsAg expressed in yeast) – HPV (papilloma L1 expressed in yeast) VIRUS LIKE PARTICLES 71 Recombinant Advantage – Safe. Growth in non pathogenic yeast cells – Easier – in case of difficult to grow viruses like hepB, HPV. Disadvantages: – Need to identify protective antigen/s – Obtaining antigen in 'correct' conformation – Usually poorly immunogenic alone – Poor CMI – requires adjuvant. 72 Hepatitis B 73 Hepatitis B 74 Hepatitis B More than 2 billion people alive have at some time been infected with Hepatitis B virus. 350,000 remain chronically infected carriers. Every year there are – 4 million acute cases – 1 million deaths Carriers – 25% of children (<7 years) become carriers. – 10% of older children and adults become carriers. 75 Aims of Hep B Vaccination Vaccination aimed at reduction of: – Clinical disease. – HBV transmission. – Chronic hepatitis and its associated liver damage and liver cancer. 76 Hepatitis B During infection large amounts of HBsAg are produced. Only small amounts of HBsAg combine with the cores to form whole virus. Remainder is released into the blood stream as spherical particles or filaments. 77 Immune Response to Infection Antibody to HBsAg indicates clinical recovery and is associated with immunity to Hepatitis B. Antibody to HBcAg is also produced and is first to appear after infection. – Present in blood of acute and chronic subjects as well as those recovered. – This antibody does not neutralize virus. 78 Plasma derived Vaccines 79 Plasma derived Vaccines Safe and Effective. Rely on supplies of infected plasma. Long production cycles. Fears of contamination with other blood borne diseases. 80 Recombinant Vaccines E.coli S. cerevisiae 81 Recombinant Vaccine 82 Recombinant Vaccine The gene coding for HBsAg was discovered in 1970. The gene has been inserted into a yeast cell. As the yeast cell grows it produces large amounts of HBsAg. The HBsAg is extracted and purified then incorporated into the vaccine. 83 Recombinant Vaccine Advantages of the recombinant vaccines. – Produced more quickly. – In larger quantities. – Free from infectious virus particles. 84 Recombinant DNA modified organisms Live Vectors Cloning of genetic material from one organism into another. The non virulent parent organism expresses the antigens of the cloned genetic material. A vaccine would elicit a response against the introduced antigen as well as the original organism. 85 Recombinant DNA modified organisms Vaccinia virus expressing papilloma virus antigens on its surface. 86 DNA Vaccines Involves the injection of naked DNA coding for one or more genes. The gene is grafted onto another piece of DNA which acts as a vector. Injected into muscle tissue, once in the cell the gene prompts the cell to produce antigen. The immune system then mounts an immune response. 87 DNA Vaccines Viral protein mRNA Antibody-producing cell Nucleus Injected DNA Class1 MHC coding for a specific antigen Cytotoxic T-lymphocyte 88 DNA Vaccines Clinical trials to date with naked DNA vaccines have not proved to be that successful DNA vaccines may be useful as a priming dose in prime-boost regimes due to their ability to induce cell mediated immune responses. 89 Thank you 90