1. DESIGN AND PRODUCTION OF RECOMBINANT SUBUNIT VACCINES BY Dr. Ahmad Usman Zafar National Centre of Excellence in Molecular Biology, Lahore

2. Vaccination is the most cost-effective method of reducing animal suffering and economic losses due to infectious diseases in animals

3. The advancement of genomics, proteomics and biotechnology provide us the opportunity to develop safe and more effective vaccines

4. ACTIVE VACCINE Stimulates Humoral Immune Response,Cellular Immune Response or Both, with the aim of protecting against or eliminating a pathogen PASSIVE VACCINE Preparation of Abs, Protect against a pathogen or disease and is administered before, at or around the time of known or potential exposure

5. comparison of different vaccine types LIVE VACCINES (ATTENUATED) (MMR, Oral Polio) Advantages:  One or few doses required  Long lasting protection  Both humoral and cellular responses Disadvantages:  Controlled attenuation normally required  Poorly defined composition  Risk of reversion to pathogenicity  Certain risk of transmission

6. comparison of different vaccine types KILLED VACCINE ( Polio and Influenza) Advantages:  No risk of reversion to pathogenicity  No risk of transmission Disadvantages:  Multiple dose typically required  Poorly defined composition  Antigen produced by cultivation of a pathogen  Mainly humoral responses  Adjuvants normally needed

7. comparison of different vaccine types TOXOID ( Tetanus and Diphtheria) Advantages:  Product is devoid of live organism  Implies greater safety Disadvantages:  Multiple dose typically required  Relatively expensive to manufacture  Cultivation of a pathogen for toxin production

8. SUBUNIT VACCINES SUBUNIT VACCINES ARE DEFINED AS THOSE CONTAINING ONE OR MORE PURE OR SEMI-PURE ANTIGENS

9. comparison of different vaccine types SUBUNIT VACCINES (NON-RECOMBINANT) Constituent proteins of bacteria or virus are isolated and purified Advantages:  Defined Composition  Various delivery systems available Disadvantages:  Antigens must be produced and purified by cultivation of a pathogen  Multiple doses typically required  Adjuvant needed

10. RECOMBINANT SUBUNIT VACCINES Identify and isolate a specific gene from virulent bacteria or virus (gene that codes immuno protective protein). Gene is inserted into plasmid DNA and ligated with ligase. New (engineered) plasmid inserted into another bacterium (transform). Allowed to grow and actually produce the antigenic protein. The vaccine is comprised of purified proteins recovered from the expression vector. Target gene

11. comparison of different vaccine types RECOMBINANT SUBUNIT VACCINES Advantages:  No risk of pathogenicity  Defined composition  Various delivery systems  Simplified large scale production  Further engineering possible Disadvantages:  Multiple doses typically require  Adjuvants needed

12. comparison of different vaccine types RECOMBINANT VECTOR VACCINES.  Based on microorganisms such as viruses or bacteria that do not cause disease in target animals or humans.  The viruses or bacteria are used as vectors, or carriers, to deliver harmless genes into the cells of the body.  The body produces proteins from the genes and these proteins stimulate an immune response against the specific protein.

13. comparison of different vaccine types RECOMBINANT GENE DELETED VACCINES Involves isolation and removal of viral gene(s) that code for “non- required” proteins This process is intended to decrease the virulence of the virus making it suitable for administration in vaccine Advantages:  The absence of specific antigens from the virus can be used to differentiate between vaccine virus and natural (“wild-type”) virus. Disadvantages:  Potential exists for virus to revert to its original, virulent state.  Degree of protection could be limited since immune response is restricted to antigens present on the virus.

14. comparison of different vaccine types RECOMBINANT VIRAL-VECTOR VACCINE Isolate an immunoprotective protein gene from a virulent virus. Clone the gene to a vector of a non virulent virus. These live vectored vaccines are being used to not only control infectious diseases of domestic animals, but of wildlife as well. This approach has resulted in a dramatic reduction in transmission of RABIES from wildlife to domestic animals and humans. This would not have been possible by conventional methods.

15. comparison of different vaccine types RECOMBINANT VIRAL-VECTOR VACCINE Advantages: Risk of reversion to virulence is eliminated if virus vector is not capable of intracellular replication. Both CMI and Humoral Immunity is good if the vector is capable of intracellular replication. Disadvantages: Weak CMI response if vector is incapable of intracellular replication. The vaccine utilizes very specific protective proteins. The immune response may be reduced in some animals.

16. recent development in vaccinology POLYNUCLEOTIDE VACCINATION This technology has been referred to as genetic immunization or DNA immunization The basis for this approach to immunization is that cells can take-up laboratory made DNA and express the genes within the transfected cells Thus, the animal acts as a bioreactor to produce the vaccine

17. recent development in vaccinology ADVANTAGES OF POLYNUCLEOTIDE IMMUNIZATION  Safe and long lived immunity  inexpensive  can induce immune responses in the presence of maternal antibodies Most recently, it has also been used for immunizing fetuses. Thus, animals are born immune to the pathogens and have life long protection Also being tested in humans against malaria, influenza, and HIV

18. TARGETS FOR THE DEVELOPMENT OF SUBUNIT VACCINE – Identifying genes – Isolating genes – Modifying genes – Re-expressing genes in other hosts or organisms

19. Recombinant Protein Expression Systems Bacteria (Escherichia coli) Yeast (Pichia pastoris) Virus (Baculovirus) Animal cell culture (CHO) Plants Sheep/Cows

20. Bacterial Systems Grow quickly (8-12 hrs to produce protein) High yields (50-500 mg/L) Low cost of media (simple media constituents) Low fermentor costs Difficulty expressing large proteins (>50 kD) No glycosylation or signal peptide removal Eukaryotic proteins are sometimes toxic Can’t handle S-S rich proteins Advantages Disadvantages

21. Yeast Systems Grow quickly (12-24 hrs to produce protein) Very high yields (50- 5000 mg/L) Low cost of media (simple media constituents) Low fermentor costs Can express large proteins (>50 kD) Glycosylation & signal peptide removal Has chaperonins to help fold “tough” prtns Can handle S-S rich proteins Advantages More advantages

22. Baculovirus Systems Advantages Disadvantages Grow very slowly (10-12 days for set-up) Cell culture is only sustainable for 4-5 days Set-up is time consuming, not as simple as yeast Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal Has chaperonins to help fold “tough” proteins Very high yields, cheap

23. Mammalian Systems Selection takes time (weeks for set-up) Cell culture is only sustainable for limited period of time Set-up is very time consuming, costly, modest yields Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal, generates authentic proteins Has chaperonins to help fold “tough” proteins Advantages Disadvantages

24. DEVELOPMENT OF RECOMBINANT HBV SUBUNIT VACCINE

28. Marker +VE -VE HB6 HB7 HB14 HB15 HB19 PCR AMPLIFICATION OF PRE-S1 REGION 1.4 Kb

29. PCR confirmation of T-A clones EcoR1 Restriction Digestion for confirmation of T-A clones

30. PCR confirmation of positive clones Vector product without insert  PCR product  PCR Confirmation of HBsAg Positive Clones

31. Expression optimization of HBsAg in P.pastoris M S14 S15 S16 S21 S25 GS115 M 25 kDa

32. Western Blotting With Anti-HBsAg kDa 115 96 65 50 35 25 15 10 1 2 3 -Ve M 15% SDSPAGE Western Blot 25kDa HBsAg Induced

33. Elisa With Anti-HBsAg PBS +Ve Recombinant clone with HBsAg GS115(Host)

34. PRODUCTION OF SUBUNIT VACCINE SEED CULTURE BANK FERMENTATION HARVESTING CELL LYSIS PROTEIN SOLUBLIZATION / REFOLDING FILTRATION / CONCENTRATION PROTEIN PURIFICATION FORMULATION PACKAGING / QC MARKETING

35. THANKYOU Biotechnology offers new approaches to animal production and health which could benefit the region