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Production Group

This article discusses the requirements, current strategies, and limitations of HIV vaccine production. It covers topics such as inactivated or killed vaccines, live attenuated vaccines, subunit vaccines, and the development of the full-length envelope glycoprotein.

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Production Group

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  1. Production Group • Vaccine Production by Rodjana Chunhabundit • Interleukin-2by Prasit Faipenkhong • Waste Water Treatment for IL-2 Production Plantby Wang Dong Mei • Plant Location and Quality Control by Waraporn Pornlab • Design of New Protease Inhibitors and RitonavirSynthesisby Sathaporn Prutipanlai

  2. Production Group • Purification Strategiesby Thida Chanyachukul • Production Plant Designby Udomsak Kongmung • Pollution Control Strategiesby Kanatip Ratanachoo • Production Cost Analysisby Siranee Sreesai

  3. HIV Vaccine Production Rodjana Chunhabundit Toxicology Program, Faculty of Science Mahidol University

  4. Outlines • Introduction - Requirement and strategies for HIV vaccines • Contents - Implementation of candidate HIV vaccine - Production techniques - Clinical trials of candidate HIV vaccines • Conclusion

  5. The Need for HIV Vaccine • The high infection rate • The high cost of palliative care and drug therapies • Poor toleration of drugs and the emergence of drug resistance • Persistent of virus in the host To stop the global HIV/AIDS pandemic

  6. Requirements for an HIV vaccine The successful vaccine must induce • Neutralizing antibody • Lymphokines • Cell mediated immunity (CMI) CTLs • Long-lasting memory T and B cells

  7. Current Strategies for an HIV Vaccine • Inactivated or killed vaccines • Live attenuated vaccines • Subunit vaccines • Recombinant envelope protein • Peptide vaccine • Live vector-based vaccines • DNA vaccines (DNA plasmids)

  8. Inactivated or Killed Vaccines • Whole viruses killed or inactivated by some chemicals. • Antigens are presented in a fashion similar to the way they are presented by real pathogen. • Incomplete activation of HIV viruses can lead to inadvertent infection of vaccines.

  9. Preparation of the inactivated whole-virus vaccine derived from a proviral DNA clone of SIV I. Preparation of the infectious SIV • Transfecting a proviral DNA clone of SIV from sooty mangbeys into CEM x174 cells • Collect supernatants from expanded cultured, clarify and concentrate 100 fold by ultrafiltration • Purify concentrated virus by ultracentrifugation through a 20% glycerol • Resuspened pelleted, partially purified virus in phosphate-buffer saline and store at -70oC until inactivation

  10. Preparation of the Inactivated Whole-virus Vaccine Derived from a Proviral DNA Clone of SIV II. Inactivation of virus • Add the solution of psoralen (trioxsalen) to conc.virus at room temp. • Expose the suspension of virus to UV light (365 nm) for 15 min. • Repeat psoralen/UV light treatment for 3 times. • Dilute inactivated virus in buffer, aliquot and store in liq. Nitrogen. • Confirm inactivation by inoculating CEMx174 cells in culture.

  11. Live Attenuated Vaccines • Nonpathogenic organisms that have been engineered to carry and express antigenic determinants from pathogenic agents or pathogenic organisms in which the virulent genes have been modified or deleted. • Antigenic determinant exist in a conformation that is very similar form of the antigen in the disease-causing organisms sustained stimulus to the HIR and CIR. • An attenuated strain of HIV,mutant lacks a large of nef gene long-term non-progressors. • Live attenuated viruses may revert to pathogenic strain or cDNA from vaccine strain may enhance the cellular oncogene.

  12. Strategy for Deleting Part of the Cholera Toxin A1 Peptide DNA Sequence

  13. Limitations of Traditional Vaccines • Require animal cell culture which is expensive • The yield and rate of production are often quite low, thereby making vaccine production costly • Extensive safety precautions are necessary • Insufficiency killing or attenuation can introduce virulent organisms into vaccine spreading dis. • Attenuated strains may revert • Not all diseases (e.g., AIDS) are preventable through the use of traditional vaccines

  14. Subunit Vaccines • Components of a pathogenic organism. • Using of recombinant DNA technology. • Advantages: stable, safe, defined chemically and free from contaminate proteins and nucleic acids. • Disadvantages: costly, altered conformation fo antigenic determinants.

  15. Evidence Supporting gp 160 is the one of the candidate HIV vaccine • Immunization with gp 120 or recombinant vaccinia virus coding for the HIV gp 160 induce T-cell mediated IR. • Neutralizing epitopes are presented on both gp 120 and gp 41 proteins. • Immunization in chimpanzees with purified gp 120 has failed to protect against infection with HIV-1.

  16. Development of the Full-length Envelope Glycoprotein • Approach by cloning and expression of env. glycoproteins in bacterial, mammalian and insect cell systems. • A gp 160 expression system based on coinfection of Vero cells with two recombinant vaccinia viruses which are the vector for genes required in synthesis of env. glycoproteins.

  17. Large-scale Production of a Vaccine Recombinant Derived HIV-1 gp 160 • Construction of Plasmids - gp 160 gene flanked by bacteriophage T7 promotor and terminate sequence. - T7 RNA polymerase gene under the vaccinia promotor • Construction of Vaccinia Virus Recombinants - Infecting CV-1 cells with vaccinia virus and transfecting them with precipitate plasmid DNA. - Harvest the cells and isolate recombinant virus.

  18. Production of Recombinant Virus Stocks Vero cells + recombinant viruses 37o C, 2-3 days shaken and centrifuged supernatant cells stored at 4o C washed and trypsinized pooled and aliquoted, frozen at -80o C

  19. Large-scale Cultivation of Vero Cells Vero cell inoculum Roller bottles 6 liter fermenter 40 liter vessel Cells adhere on microcarriers

  20. gp 160 Production 5 x 109 cells/liter medium settled recomb. virus microcarrier + medium 40 hrs detached cells pump out with the medium adhering cells are washed and rapid stirring 70 mm sieve centrifugation pellet store at -80o C

  21. Peptide Vaccines • Target epitopes most immunogenic • Peptide sequences strongly binding with neutralizing antibody • amino acid 735 to 752 of gp160, RP-35 of gp120, core protein p17 • highly specific, inexpensive and safe • tertiary structure different from neutral protein

  22. Preparation of the Hybrid T1-SP10 Peptide • Synthesize peptide by using of peptide synthesizer • Deprotect and cleave peptides from the supporting resin with hydrogen fluoride in 10% anisole • Solubilized in 15-25 % (v/v) glacial acetic acid and lyophilized • Reconstitued in PBS and dialyzed or HPLC purified • Determine molecular mass by mass spectrometry

  23. Live Vector-Based Vaccine • Live virus vectors containing HIV gene • Sustained expression of large amount of HIV Ag neutralizing Ab and CTLs responses • Vaccinia virus is a strong candidate as a vector vaccine because • It can express inserted gene independently of host regulatory or enzymatic functions • broad host range, stable, benign virus • Gene coding for specific antigen must be introduced into viral genome by in vivo homologous recombination

  24. Method for the Integration into Vaccinia Virus of a Gene Whose Protein Product

  25. DNA Vaccines • In vivo delivery of antigen-encoding plasmidDNAs, not protein immunogens • Correctly folded and glycosylated antigens in vivo effective HIR and CIR against live virus challenge • Cloned gene encoding antigen in a plasmid is coated on a gold particles • ‘Gene gun’ is used to accelerate DNA-coated gold particles into target tissue

  26. Clinical Trials of HIV Vaccines Phase I trial - evaluate safety and immunogenic response - sample size and the length of follow-up are grater than other vaccines - population for phase I trial should be individuals at no risk of HIV infection Phase II trial - determine optimal dosage schedules based on safety and immunogenicity data - larger number of volunteers - high and low-risk populations

  27. Phase III trial -placebo-controlled, randomized and double-blind studies - number of volunteers is determined by incidence of infection rate and statistical parameters of protection - be performed only in the most promising candidate vaccines - target populations, including Homosexual men, intravenous drug users, prostitutes, prisoners, newborn of seropositive mother, and seual transmitted diseasepatients

  28. Phase of Clinical Trials of a Candidate AIDS Vaccine Phase Type of Duration Purpose subjects 1 Low risk Up to 1 yr Mainly safety, some immunogenicity 2 Low & high Up to 2 yr Safety, immunogenicity risk 3 All target Up to 4 yr Efficacy, safety, populations dosage

  29. AIDS Vaccines Currently in Clinical Trials • Subunit Vaccines - Recombinant envelope protein: gp 160, gp 120 - Peptide: V3 peptide, T-B peptide • Live vector-based vaccines - Vaccinia envelope - Canarypox envelope • DNA vaccines

  30. Conclusion The preventive HIV vaccine - elicit specific CTLs and neutralizing antibody for all strains of HIV - easy administration - safe - low cost

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