5. vaccine technology


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5. vaccine technology

  1. 1.  A vaccine is any preparation of dead or attenuated pathogens, or their products, that when introduced into the body, stimulates the production of protective antibodies or T-cells without causing the disease
  2. 2.  The terms vaccination and vaccine derive from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows; vacca, cow) protected them from the highly contagious and frequently fatal disease smallpox He tested his theory in 1796 by inoculating 8-year-old James Phipps with liquid from cowpox pustule Subsequent inoculation of the boy with smallpox produced no disease Since Jenners time, the term has been retained for any preparation intended to attain the same
  3. 3. Edward Jenner (1749-1823)
  4. 4.  However, this approach dates back, well before Jenner’s time in India and China where they performed what is known as variolation  Variola virus=smallpox virus Variolation, a procedure developed in China and India 1000 AD used a live smallpox vaccine to generate immunity Employing several different techniques ‘well individuals’ were exposed to variolous material from a human with a milder form of smallpox—presumably in the expectation that this would cause less severe disease in the recipient—an early form of ‘attenuation
  5. 5.  Whole organism  Live/attenuated vaccines  Killed/inactivated vaccines Toxoids Peptide vaccines Recombinant vaccines DNA vaccines
  6. 6.  Live/attenuated vaccines make up the bulk of successful viral vaccines Are prepared from attenuated strains that are almost or completely devoid of pathogenicity but are still immunogenic They multiply in the human host and provide continuous antigenic stimulation over a period of time
  7. 7.  Use of a related virus from another animal – e.g. the use of cowpox to prevent smallpox Administration of pathogenic or partially attenuated virus by an unnatural route  the virulence of the virus is often reduced when administered by an unnatural route  immunization of military recruits against adult respiratory distress syndrome using enterically coated live adenovirus type 4, 7 and (21).
  8. 8.  Passage of the virus in an "unnatural host" or host cell –  the major vaccines used in man and animals have all been derived this way  After repeated passages, the virus is administered to the natural host  The initial passages are made in healthy animals or in primary cell cultures  Examples: the 17D strain of yellow fever (in mice and then in chick embryos), Polioviruses (in monkey kidney cells) and measles (in chick embryo fibroblasts) Development of temperature sensitive mutants  this method may be used in conjunction with the above method
  9. 9.  The vaccine is injected sc/im, virions enter various cell types (APCs) using receptor- mediated endocytosis Proteolytic degradation of viral proteins occurs, the peptides produced are then loaded onto MHC I molecules The complex is displayed on the cell surface Circulating cytotoxic T cells recognize the complex, become activated and and release cytokines
  10. 10.  The cytokines trigger apoptosis (programmed suicide) of the infected cells Some Tc become memory cells but the basis of this is incompletely understood Additionally, immature DCs will phagocytose virus vaccine initiating a series of events that leads to the production of plasma cells, neutralizing IgG antibodies and memory B cells
  11. 11.  One important advantage of live/ attenuated vaccines is that they are sufficiently immunogenic therefore primary vaccine failure are uncommon and are usually the result of inadequate storage or administration
  12. 12.  Several potential safety problems exist with live/attenuated vaccines:  Underattenuation  Mutation leading to reversion to virulence  Preparation instability  Contaminating viruses in cultured cells  Heat lability  administration to immunocompromized or pregnant patients may be dangerous
  13. 13.  The term killed generally refers to bacterial vaccines whereas inactivated relates to viral vaccines An inactivated whole organism vaccine uses pathogens which are killed and are no longer capable of replicating within the host The pathogens are inactivated by heat or chemical means while assuring that the surface antigens are intact Inactivated vaccines are generally safe, but are not entirely risk free
  14. 14. Organism Method of inactivationRabies β-propiolactoneInfluenza β-propiolactonePolio FormaldehydeHepatits A formaldehyde
  15. 15. Organism Method of inactivationSalmonella typhi Heat plus phenol or acetoneVibrio cholera HeatBordetella pertusis Heat or formaldehydeE.Coli (experimental) ColicinYersinia pestis Formaldehyde
  16. 16.  Following injection, the whole organism is phagocytosed by immature dendritic cells Processed peptides will be presented on the cell surface as separate MHC II:antigenic fragment complexes Th2, each with a TCR for a separate antigenic fragment will be activated B cells, each with a BCR for a separate antigenic fragment will bind antigens that drain along lymph channels
  17. 17.  The separate antigens will be internalized and presented as an MHC II:antigenic fragment This will lead to linked recognition with the appropriate Th2 Activated Th2 will release IL2, IL4 IL5 and IL6, inducing B-cell activation, differentiation and proliferation with subsequent isotype switch (IgM to IgG) and memory B cell formation.
  18. 18.  First, they are safe because they cannot cause the disease they prevent and there is no possibility of reversion to virulence Second, because the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals Third, they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines are used out in the community Fourth, all the antigens associated with infection are present and will result in antibodies being produced against each of them
  19. 19.  Contamination by toxins or chemicals Allergic reactions  Surface endotoxins on inactivated pertussis vaccine occasionally induce DTH responses, and influenza virus has been linked to similar reactions, though this may be due more to the immunogenicity of the egg whites in which the virions are raised Autoimmunity
  20. 20.  Also, inactivated vaccines do not always induce protective immunity.  Multiple boosters and an adjuvant are usually necessary for continual antigen exposure  the dead organism is incapable of sustaining itself in the host, and is quickly cleared by the immune sysytem Furthermore, inactivated vaccines are generally capable of inducing humoral immunity rather than cellular immunity.
  21. 21.  A toxoid is a chemically or physically modified toxin that is no longer harmful but retains immunogenicity Certain pathogens cause disease by secreting an exotoxin:  these include tetanus, diphtheria, botulism and cholera In addition, some infections, for example pertussis, appear to be partly toxin mediated Specific physical or chemical modification of the toxins produces a toxoid, which is a vaccine
  22. 22.  The principal toxin is tetanospasmin Binds to specific membrane receptors located only on presynaptic motor nerve cells Internalization and migration of this toxin to the CNS blocks the metabolism of glycine which is essential for the normal functioning of gama amino butyric acid (GABA) neurons GABA neurons are inhibitory for motor neurons Their non-functioning results in excess activity in motor neurons This gives rise to muscle spasms, a characteristic feature of tetanus
  23. 23.  Manufactured by growing a highly toxigenic strain of Clostridium tetani in a semi-synthetic medium Bacterial growth and subsequent lysis release the toxin into the supernatant Formaldehyde treatment converts the toxin to a toxoid by altering particular amino acids and inducing minor molecular conformational changes
  24. 24.  Ultrafiltration then removes unnecessary proteins residues The toxoid is physicochemically similar to the native toxin thus inducing cross-reacting antibodies But, the changes induced by formaldehyde treatment render it non-toxigenic
  25. 25.  Upon administration (sc/im) the toxoid molecules are taken up at the vaccination site by immature dendritic cells Within this cell, they are processed through the endosomal pathway where they are bound to MHC II molecules The MHC II:toxoid complex then migrates to the cell surface mature DCs migrate along lymph channels to the draining lymph node There , they encounter naïve Th 2 cells Identifying and then binding of the MHC II:toxoid to the specific Th2 receptor then activates the naive T cell, causing it to proliferate
  26. 26.  Some toxoid molecules not taken up by DCs pass along lymph channels to the same draining lymph nodes There, they come into contact with B cells Binding to the B cell through the specific immunoglobulin receptor that recognizes the toxoid The toxoid is internalized, processing through the endosomal pathway and presented on the cell surface as an MHC II:toxoid complex as happens in the DCs
  27. 27.  These two processes occur in the same part of the lymph node The B cell with the MHC II:toxoid complex on its surface now comes into contact with the activated Th2 whose receptors are specific for this complex This process is called linked recognition The Th2 activates the B cell to become a plasma cell with the production initially of IgM, and then there is an isotype switch to IgG In addition, a subset of B cells becomes memory cells
  28. 28.  The rationale of toxoid vaccination is production of antibodies with enhanced capacity to bind the toxins They thus form complexes with the toxins preventing then to interact with toxin receptors on the nerve cells (tetanus) This is referred to as neutralization of the toxins by antibodies
  29. 29.  There are three principal advantages:  First, they are safe because they cannot cause the disease they prevent and there is no possibility of reversion to virulence.  Second, because the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals.  Third, they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines are used out in the community
  30. 30.  First, they usually need an adjuvant and require several doses (otherwise less immunogenic) Second, local reactions at the vaccine site are more common—this may be due to the adjuvant or a type III (Arthus) reaction  The reaction generally starts as redness and induration at the injection site several hours after the vaccination and resolve usually within 48–72 h  The reaction results from excess antibody at the site complexing with toxoid molecules  activating complement by the classical pathway causing an acute local inflammatory reaction
  31. 31.  A recombinant vaccine contains either a protein or a gene encoding a protein of a pathogen origin that is immunogenic and critical to the pathogen function The vaccine is produced using recombinant DNA technology The vaccines based on recombinant proteins are also called subunit vaccines  e.g. RTS,S malaria vaccine, passed phase II now entering phase III
  32. 32.  The logic of such vaccines, in simple terms, is as follows:  Proteins are generally immunogenic, and many of them are critical for the pathogenic organism  The genes encoding such proteins can be identified and isolated from a pathogen and expressed in E. coli or some other suitable host for a mass production of the proteins  The proteins of interest are then purified and mixed with suitable stabilizers and adjuvants, if required, and used for immunization
  33. 33.  The first step is to identify a protein that is both immunogenic and critical for the pathogen The gene encoding this protein is then identified and isolated The gene is integrated into a suitable expression vector and introduced into a suitable host where it expresses the protein in large quantities The protein is then isolated and purified from the culture system It is used for the preparation of vaccine
  34. 34. 1. Genetically engineered microorganisms, e.g., yeast for the expression of hepatitis B surface antigen (HBsAg) used as vaccine against hepatitis B virus2. Cultured animal cells, e.g., HBsAg expressed in CHO (Chinese hamster ovary) cell line and C-127 cell line4. Transgenic plants, e.g., HBsAg, HIV-l (human immunodeficiency virus-I) epitope (in experimental stages)5. Insect larvae; the gene is integrated into a bacculovirus genome, which is used to infect insect larvae. Often a very high quantity of the recombinant protein is produced .
  35. 35.  An alternative application or recombinant technology is the production of hybrid virus vaccines e.g. HBsAg vaccine Here, vaccinia virus is used as a carrier for genes that encode antigenic proteins of interest The genes may be derived from organisms which are difficult to grow or inherently dangerous, and the constructs themselves are replication deficient, nonintegrating, stable and relatively easy to prepare
  36. 36.  DNA sequence coding for the foreign gene is inserted into the plasmid vector along with a vaccinia virus promoter and vaccinia thymidine kinase sequences The resultant recombination vector is then introduced into cells infected with vaccinia virus to generate a virus that expresses the foreign gene The recombinant virus vaccine can then multiply in infected cells and produce the antigens of a wide range of viruses The genes of several viruses can be inserted, so the potential exists for producing polyvalent live vaccines
  37. 37.  Proteins encoded by these genes are appropriately expressed in vivo with respect to glycosylation and secretion They are processed for major histocompatibility complex (MHC) presentation by the infected cells, thus effectively endowing the host with both humoral immunity and CMI
  38. 38.  Small peptide sequences corresponding to important epitopes on a microbial antigen can be synthesized readily economically Some long ones are also being invented, but are more expensive to manufacture  eg. Pf MSP-3 long synthetic peptide (now in phase II)
  39. 39.  Synthetic peptides can be highly immunogenic in their free form provided they contain, in addition to the B cell epitope, T- cell epitopes recognized by T-helper cells The T-cell epitope must be linked to the B-cell epitope Such T-cell epitopes can be provided by carrier protein molecules, foreign antigens or within the synthetic peptide molecule itself
  40. 40.  The antigens are precisely defined and free from unnecessary components which may be associated with side effects They are stable and relatively cheap to manufacture Furthermore, less quality assurance is required Feasible even if the pathogen cannot be cultivated Changes due to natural variation of the virus can be readily accommodated
  41. 41.  DNA vaccines are usually circular plasmids (supercoiled) that include a gene encoding the target antigen (or antigens) under the transcriptional control of a promoter region active in human cells With DNA vaccines, the subject is not injected with the antigen but with DNA encoding the antigen
  42. 42.  DNA vaccines are composed of a bacterial plasmids Expression plasmids used in DNA-based vaccination normally contain two units:  Antigen expression unit composed of promoter/enhancer sequences followed by antigen- encoding and polyadenylation sequences  The production unit composed of of bacterial sequences necessary for plasmid amplification and selection The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology
  43. 43.  Sometimes DNA sequences encoding  costimulatory molecules  sequences that target the expressed protein to specific intracellular locations (e.g., endoplasmic reticulum) Once constructed, the vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from the much larger bacterial DNA and other bacterial impurities
  44. 44.  This purified DNA acts as the vaccine The DNA vaccine can be injected into a muscle just as conventional vaccines are  Using ordinary syringe or gene gun DNA vaccines elicit cell-mediated as well as antibody-mediated immune responses
  45. 45.  A plasmid vector that expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter is injected into the skin or muscle of the the host After uptake of the plasmid, the protein is produced endogenously The protein is processed intracellularly into small antigenic peptides by the host proteases and presented to the cell surface with MHC I Subsequent CD8+ cytotoxic T cells (CTL) are stimulated and they evoke cell-mediated immunity
  46. 46.  CTLs inhibit viruses through both cytolysis of infected cells and noncytolysis mechanisms such as cytokine production The foreign protein can also be presented by the MHC class II pathway by APCs which elicit helper T cells (CD4+) responses Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated to produce antibodies production This is the same manner in which traditional vaccines work
  47. 47.  The plasmid is taken up by an antigen- presenting cell (APC) like a dendritic cell The gene(s) encoding the various components are transcribed and translated The protein products are degraded into peptides These are exposed at the cell surface nestled in class I histocompatibility molecules MHC I-peptide complex serves as a powerful stimulant for the development of cell-mediated immunity
  48. 48.  If the plasmid is taken up by other cells (e.g. muscle cells) The proteins synthesized are released and can be engulfed by antigen-presenting cells (including B cells) In this case, the proteins are degraded in the class II pathway and presented to helper T cells These secrete lymphokines that aid B cells to produce antibodies
  49. 49.  So far, most of the work on DNA vaccines has been done in mice where they have proved able to protect them against tuberculosis, SARS, smallpox, and other intracellular pathogens Different DNA vaccines against HIV-1 — the cause of AIDS — are in clinical trials
  50. 50.  Delivery of the DNA to cells is still not optimal, particularly in larger animals The possibility that exists with all gene therapy, that the vaccines DNA will be integrated into host chromosomes and will  either turn on oncogenes  or turn off tumor suppressor genes Antibiotic resistance? Extended immuno-stimulation by the foreign antigen could in theory provoke chronic inflammation or autoantibody production  
  51. 51.  Thousands of individual steps (summary)  Recognize the disease and Identify the etiologic agent  Attempt to grow the agent in laboratory  Establish an animal model for disease  Identify an immunologic correlate for immunity to the disease- Usually a serum antibody  choose antigen (in laboratory)  Prepare candidate vaccine following Good Manufacturing Practice
  52. 52.  Evaluate candidate vaccine for ability to protect animal model Prepare protocol for human studies Phase I human trials- Safety and immunogenicity using small group (phase Ia in company area) Phase II trials- Safety and immunogenicity using relatively larger group Phase III trial- Efficacy and safety Long and complicated process  Usually takes 10–15 years  Many vaccine candidates fail for every success
  53. 53. The vaccine:• Attenuated organisms revert to wild type (e.g. polio types 2,3)• ‘Killed’ organisms not properly killed (has happened with polio)• Inclusion of toxic material (e.g. typhoid, pertussis)• Contamination by animal viruses• Contamination by egg proteins (hypersensitivity)• Cross-reaction with ‘self’ (autoimmunity)The patient:• Immunodeficiency (attenuated organisms may cause serious/fatal disease)• Local inflammatory reactions, often to the adjuvant• Worsening of disease by increasing immunopathology (risk of therapy)• Hypersensitivity to vaccine (e.g. tetanus)• Interference between vaccines given together (not always)• Induction of inappropriate response (e.g. dengue)
  54. 54. •Public aversion to the risks of adverse effects pushes towards sub-unit vaccines rather than live attenuated •Pathogens change their antigens (or have distinct life cycle stages) a) makes initial composition of the vaccine components complicated b) make a successful vaccine redundant eg. antigenic drift/shift in influenza, (HIV) •Success so far has been where naturally occurring immunity is strong eg childhood viruses BUT this is not the case for HIV, TB, malaria etc –so vaccine has to be even better •Successes to date mostly with antibody inducing vaccines- not cell mediated! albendazole•Confounding effects in tropical populations-eg AIDS, malnutrition, pre-existing exposureto environmental organisms (eg re BCG)- parasitic diseases (Th1 vs Th2 balances)Does deworming prior to vaccination enhance BCG vaccine?Elias et al., Clin Exp Immunol. 2001 123: 219-25
  55. 55. Summary•Early vaccines were mostly whole dead or liveattenuated•These provide their own integrated adjuvant/antigens•Now the shift is towards defined subunit-basedvaccines•This requires you to select which adjuvant/antigen to include•Immunology is now having a greater impact onvaccine design:-optimized activation (e.g. TLRsynergy)-temporary removal of immune-regulation? (e.g. anti-IL10)•Safety, efficacy and memory are the key!
  56. 56.  Peter Delves, Seamus Martin, Dennis Burton and Ivan Roitt: Roitt’s Essential immunology, 11th edition 2006 page 287-311 David Baxter. Active and passive immunity, vaccine types, excipients and licensing: Occupational Medicine 2007;57:552–556 Medical immunology 2006: edited by Gabriel Virella. ‑‑ 6th ed Goering RV, Dockrell HM, Zuckerman M, Wakelin D, Roitt IM, Chiodini PL and Mims C; Mims’ Medical Microbiology 4th Edition, Philadelphia Elsevier (2008) page 519-542 http://www.brown.edu/Courses/Bio_160/Projects1999/vaccineovervie http://www.microvet.arizona.edu/Courses/MIC419/Tutorials/vac cines.html www.google.com/(specific topic)