PEPTIDE AND SUBUNIT VACCINES

1. PEPTIDE AND SUBUNIT VACCINES

2. COURSE CONTENTS • Vaccines- Introduction • Vaccines- History • Immunological Adjuvants • Types • DNA Vaccines • Viral Vectors • Mucosal Vaccines • Peptide Vaccines • Subunit Vaccines • Vaccine Manufacturing • Vaccine Safety Issues • Animal Testing of Vaccines • HPV Vaccine- A case study of Vaccine Development

3. PEPTIDE VACCINES • Peptide Vaccine are formed by using a specific domain of an antigenic protein. • Instead of encoding whole of the protein, only the epitopic region can be added in to the vaccine preparation.

4. BASIC STEPS IN PEPTIDE VACCINE PREPARATION • Epitope/ target peptide Identification • Linking • Delivery

5. 1. EPITOPE IDENTIFICATION • Antigen based • or • Antibody based

6. 1. a. Antigen based identification • Synthetic peptides have been used as powerful tools to identify class II and class I-restricted T cell epitopes by systematic screening in functional assays • MHC-bound peptides eluted directly from tumor material of patients were identified on the basis of their mass and partial sequence by highly sensitive mass spectrometry • Pool sequencing of MHC-eluted peptides has revealed that peptides bind to MHC I via an allele-specific consensus motif of anchor residues • MHC molecules therefore encoded by different gene loci bind their own distinct (sometimes overlapping) family of peptides

7. Measles virus- epitope identification • Strategy based on peptides of the hemagglutinin (H) and fusion (F) MV neutralizing antibodies are directed against these glycoproteins • Sequential epitopes for both the proteins have been identified that have the inherent property of stimulating either B or T cell epitopes • Two sequential B cell epitopes have been identified on the H protein by screening a panel of neutralizing and protective antibodies against a complete set of overlapping synthetic peptides HA

8. EPITOPE IDENTIFICATION- CANCER CELLS • Cancer T-cell Epitopes (TCEs) • Cancer vaccines can be based on whole cancer cells*, Tumor Associated Antigens (TAAs) or peptide fragments of TAAs. • Whole cells- difficult to attain • TAA- laborious, expensive and resemble self antigens causing a problem in discrimination. • So, recombinant TAAs appear to be obvious antigens for tumor vaccines. They are: • Most TAAs are not tumor specific • Peptides derived from TAAs are the true antigens recognized by the T cells • Ease of expression as production of TAA peptides in large quantities may serve as a tumor vaccine

9. CANCER VACCINE-PREPARATION

10. 1. b. AntiBODY/ PARATOPE based identification • Antigenic Determinants on Abs Fall in 3 Categories:

11. Idiotypic network

12. 1. b. AntiBODY/ PARATOPE based identification • The idiotype (Id) i.eidiotypic determinant is associated with the hypervariable region of the antibody (Ab) molecule • It represents the unique antigenic determinant of that Ab • An Ab-1 is defined as an Ab that recognizes a particular antigen e.g a vaccine candidate • The Id on Ab-1 itself can act as an immunogen that can elicit an immune response; the Abs that bind to the Id on Ab-1 are referred to as anti-idiotypic antibodies ( anti-Id) or Ab-2 • The paratope is the site on Ab-1 that binds to a particular antigen; thus the binding site of an anti-paratope antibody is a molecular mimic of the antigen.

13. ANTI-IDIOTYPIC ANTIBODIES TO CANCER CELLS- an experiment

14. 2. linker • Organic stretches • Glycans and other sugars

15. 3. CARRIER MOLECULE • Peptides need to be linked to another molecule to prevent rapid degradation (HAPTEN). • 1. Keyhole limpet hemocyanin (KLH) • an inert carrier protein from a marine gastropos mollusk • 2. Hepatitis B core protein (HBcAg) • highly immunogenic carrier protein which self assembles into small particles

16. Limitations of Peptide Vaccines • Epitope must consist of a contiguous stretch of amino acids • Not all peptides are effective in eliciting an immune response (may need two or more) • Peptide must have the same conformation as in pathogen • Amount of peptide required to elicit an immune response may be 1000X more than for inactivated pathogen

17. SUBUNIT VACCINES • Subunit vaccines consist of one or more antigens purified from the microorganism or produced by recombinant DNA technology or chemical synthesis

18. processing • Development of subunit vaccines requires knowledge of: • Protective antigen(s) • Ability to produce and purify those antigen(s) on large scale • Ability to prove their protective efficacy in appropriate animal models in vivo and/or in vitro assays • Identification of the potential candidates for development of subunit vaccines therefore has to be based on approach combining study of: • Genetics • Biochemistry • Immunology

19. PROTEIN BASED • The identification of protective antigens is a complex problem and involves approaches that can differ for viruses, bacteria and parasites • Viruses have generally small genomes encoding a few proteins that can easily be selected when compared with larger microorganisms • Envelope proteins and glycoproteins are the primary candidates for the induction of neutralizing antibodies whereas core antigens are usually good candidates for CTL responses • For bacteria and parasites there can be several hundred potential candidate antigens

20. CARBOHYDRATE BASED • The first subunit vaccines developed have been the diphtheria and tetanus toxoids • Semi-purified toxins are inactivated by chemical (formaldehyde) treatment and used as vaccines • For these two vaccines the titers of serum antitoxin antibodies correlate well with the protection • For encapsulated bacteria the capsular polysaccharide as an antigen was chosen for development of vaccine on the observation that mutants without capsule are non-pathogenic • Meningococcal group specific immunity is mediated by serum antibodies directed against the group-specific capsular polysaccharide.

21. EXAMPLES • Purified capsular polysaccharides have been used to develop vaccines against • MenC, • N. meningitidis group A (MenA) • N. meningititdis group Y (MenY) • N. meningititdis group MenW135 (MenW135) • Using the polysaccharide, vaccines have been developed against • 23 serotypes of S. pneumoniae • Hib • Salmonella typhi

22. drawbacks • All these vaccines have several drawbacks • Capsular polysaccharides are T-independent antigens and they induce transient antibody responses (mostly IgM and IgG2 isotypes) in individuals aged over 18 with no efficacy in infants • Polysaccharides do not induce immunological memory • Repeated immunization not only fails to induce any increase in specific antibody titers but can also in some cases even induce tolerance (in adults) • To overcome these drawbacks the toxoids and the polysaccharides have been put together in the form of a conjugate vaccine

23. conjugation

24. POSSIBLE SOLUTIONS • During 1920s it was demonstrated that the immunogenicity of saccharides was significantly increased when animals were immunized with the sugarcovalently linked to protein which behaved as carrier molecule • This process converts T-independent antigens into T-dependent antigens • The only limitation of this technology is the overload of carrier protein that patients would receive in the situations where all conjugated vaccines use the same carrier • Carriers not only help the production of anti-oligosaccharide antibodies but also induce specific antibodies against themselves • Anti-carrier antibodies suppress the induction of anti-oligosaccharide antibodies in animals.

25. Conjugate vaccines • Hib • The first conjugate vaccine developed has been against Hib: • It has a high immunogenicity and efficacy • Induces immunological memory • Igisotype switching • Antibody affinity maturation in children aged less than 18 months • Un-conjugated polysaccharide vaccine fails to induce any of the above responses

26. Recombinant DNA approach for subunit vaccines • Identification of a relevant antigen can take several years • It is possible that antigens expressed during the infection in vivo are not equally well expressed in vitro during cultivation • Once a suitable antigen is identified most often it is expressed in genetically engineered prokaryotic or eukaryotic vectors • Although this approach has been a success in the case of viruses it has failed up till now for complex pathogens like bacteria and parasites

27. PARTICLES BASED APPROACH

28. particles • First application for Hepatitis B through expression of HBsAg gene in bakers yeast i.e S. cerevisae • Notably expression of the HBsAg gene in E.coli gave rise to HBsAg polypeptides but not of HBsAg particles • HBsAg is a virus like particle (VPL) in that its surface structure is similar to that of HBV virion • Purified yeast derived HBsAg is adjuvanted with Aluminum salts for formulation as vaccines • HBsAg has also been expressed in transgenic potato tubers (immunogenic and edible) • HPVvirion is a highly ordered structure with major protein L1 • Expression of L1 in eukaryotic cells results in the formation of VLPs that elicit HPV-neutralizing antibodies leading to protective immunity

29. Vaccines for non-infectious diseases • Allergy • Oncology • Autoimmunity • Crude extracts from allergens such as ragweed can be used as therapeutic vaccines • Therefore an allergen encoding gene is expressed in a hetrologous host cell • DIABETES • Before the clinical symptoms of the disease develop, autoantibodies become detectable to pancreatic beta cell autoantigense.ginsulin. Following which beta cells are destroyed by autoimmune attack. • A range of recombinant autoantigens can prevent the development of type 1 diabetes in a mouse model • Clinical trials show that subcutaneous injection of E.coli derived recombinant insulin into pre-diabetic patients resulted in a significant delay in development of clinical type 1 diabetes

30. HOSTS FOR THE EXPRESSION OF RECOMBINANT PROTEINS • Bacteria • E.coli • Bordetellapertusis • Vibrio cholerae • Yeast • S. cerevisiae • Hansenulapolymorpha • Mammalian cells • Chinese hamster ovary • African green monkey kidney • Lymphoid cells • Mammals • Goat • Sheep • Cow • Plants • Tomato • Potato • Tobacco

31. Failures in the development of subunit vaccines • Among the examples of failures of conventional subunit vaccines are three diseases: • Acquired Immunodeficiency Syndrome • Tuberculosis • Malaria • AIDS • Approaches to vaccine development have essentially focused onto the proteins of the HIV-1 envelope mainly gp120 • Problems: • Antigen variability • Poor ability of these vaccine constructs to induce antibodies that are able to neutralize heterologous viral strains • Effective approach is to aim for inducing both antibody and CTL responses

32. Failures in the development of subunit vaccines • Tuberculosis • Problems: • Intracellular location of Mycobacterium tuberculosis, which needs the development of Th1-type and MHC class I-restricted CTL responses • Lack of ideal experimental animal models of infection • Malaria • Problems • Immunity against malaria parasites is not only specie specific but also specific for each stage of development • The effector mechanisms required for protection are different for each developmental stage • Ideal malarial vaccine therefore should consist of a mixture of antigens from the different stages of development of the parasite

33. Thank you