hepatitis B vaccine.pptx

hepatitis B vaccine

1. Recombinant DNA Technology in VACCINE PRODUCTION HEPATITIS B SUBMITTED TO : Dr Sandip V. Pawar SUBMITTED BY: Group 2

2. INTRODUCTION The term hepatitis describes inflammation of the liver. Hepatitis may be caused by alcohols, drugs, autoimmune diseases, metabolic diseases, and viruses. Viral infections accounts for more than half the cases of acute hepatitis. Viral hepatitis is a systemic infection affecting the liver predominately with primary inflammation of the liver by any one of a heterogeneous group of hepatotropic viruses.

3. Hepatitis A (HAV) (1973) Hepatitis B (HBV) (1970) Hepatitis C (HCV) (1988) Hepatitis D (HDV) (1977) Hepatitis E (HEV) (1983) Hepatitis F-Not separate entity- Mutant of B Virus. Hepatitis G (HGV) (1995) DIFFERENT TYPES OF HEPATITIS VIRUSES ARE :-

4. Acute hepatitis (self-limited liver injury of less than 6 months C hronic hepatitis (hepatic inflammation more than 6 months. TYPES OF VIRAL HEPATITIS The severe pathological consequences of persistent Hepatitis infections include the development of chronic hepatic insufficiency, cirrhosis and hepato cellular carcinoma (HCC). In addition, Hepatitis carriers can transmit the disease for many years.

5. INFECTION / REPLICATION OF HEPATITIS B VIRUS INSIDE HEPATOCYTES 1. The virus goes and attaches to liver cell membrane. 2. Transport of Virus inside liver cell. 3. The Virus then releases its DNA and DNA polymerase inside liver cell nucleus. 4. This Hepatitis B DNA causes the liver cell to produce HBs, HBc , HBe proteins and DNA polymerase through mRNA.

6. 5. DNA polymerase causes the liver cell to make copies of Hepatitis B DNA from mRNA. 6. The cell than assembles live copies of virus. 7. The excess number of suface proteins are produced many of which stick together to form small spheres and chains which give ground glass appearence to blood samples under microscope. 8. The copies of virus are then released from liver cell membrane into blood stream and from there it can affect other liver cells.

7. Structure of Hepatitis B Virus The hepatitis B virus is 42nm in diameter and composed of 27 nm nucleocapsid core ( HBcAG ), surrounded by outer lipoprotein coat (also called envelope) containing the surface antigen ( HBsAG ) Virion is also referred to as Dane particle ( ds-tranded DNA) Core antigens located in the center ( nucleocapsid ) ▪Core antigen ( HBcAg ) ▪e antigen ( HBeAg )

8. HBsAg = surface (coat) protein HBcAg = inner core protein HBeAg = secreted protein 19

9. Hepatitis virus is a DNA virus with a remarkably compact genomic structure. It has circular partially double-stranded DNA viruses. Replication occurs by reverse transcriptase. It is small, circular, 3200 base- pair size, HBV DNA codes for four sets of viral products and has a complex, multi particle structure.

10. PRINCIPLE OF VACCINATION FOR HEPATITIS B All available HBV vaccines contain the hepatitis B envelope protein. Hepatitis B surface antigen(s) (HBsAg) is composed of three related envelope proteins which are synthesized by the alternate use of three translational start codons and a common stop codon. The HBsAg proteins include small HBs (SHBs): S Domain The middle-sized protein (MHBs): S and pre-s2 The large HBs protein (LHBs):S, pre-s2 , preS1domain The pre-S antigens seem to be important in inducing T-cell help for production of anti- HBs. Thus, T-cell recognition requires presentation to T cells of HBV antigenic determinants, which must be processed by antigen presenting cells prior to expression on the surface of T cells in association with HLA antigens.

11. Role of envelope antigens The pre-S1 domain of the large envelope protein contains a 21–47 aa sequence, important for attachment to the hepatocyte. Pre S2 has following functions: It is a proteolysis sensitive site and it has a 5–16 aa sequence which can block a human serum albumin receptor-binding site. It also has an activated protein kinase binding site. permeabilization site, which may be important in transfer of HBV particles into the cytosol. particularly pre-S1, express highly immunogenic T- and B-cell epitopes, a property which has potential applications in third generation vaccines Initial approach of vaccine production In the late 1970s, two vaccines against HBV were developed in the United States and France, both containing purified HBsAg obtained from serum of HBsAg carriers. US product contained 22 nm HBsAg particles devoid of the pre-S proteins French HBV vaccine contained additional small and inconsistent amounts of pre-S2 and pre-S1 antigens. Concerns about safety of blood products, as well as the inconsistency as a source of raw material and the advances of recombinant DNA technology led to the development of second-generation recombinant vaccines produced in yeast

12. Recombinant vaccines two recombinant vaccines : Engerix-B (SmithKline Biologicals, Belgium) RECOMBIVAX HB-Vax II (Merck & Co., USA) [22,23]. These two vaccines contain non-glycosylated SHBs p24, which must be released from the yeast during the manufacturing process

13. Novel HBV vaccines Several novel HBV vaccines were reported in the last decade, including: (a) Yeast-derived Pre-S/S vaccines (b) Mammalian cell-derived pre-S/S vaccines (c) DNA vaccines (d) Polypeptide micelle vaccine derived from HBsAg (e) Expression of immunogenic HBV peptides in vaccinia virus (f) Synthetic polypeptides containing immunogenic sur- face or core epitopes (g) Anti-idiotype vaccines (h) Oral immunization with a recombinant salmonella gene product containing HBcAg epitopes Reference: Journal of Hepatology 39 (2003) S70–S76 EUROPEAN ASSOCIATION OF THE STUDY OF THE LIVER(EASL)

14. RECOMBINANT DNA VS PLASMA DERVIED VACCINE Traditional vaccine used a weakened or killed form of a virus to force the body to develop antibodies that are strong enough to combat the virus but by using r-DNA technology the vaccine uses the surface antigen of virus that stimulate the production of protective antibodies which combat with the HB virus Plasma derived vaccine were effective but safety concern was taken in consideration but r-DNA technology is potentially safe and effective T he potential for genetic modification of live-attenuated vaccines was seen in traditional vaccine but gene deleted pathogen is seen in r-DNA  In most instances, purified R-DNA vaccines should be more stable than comparable traditional vaccines, particularly with regard to temperature requirements. Antibody concentration were significantly higher in the group of recombinant vaccine. Therefore this vaccine has superior immunogenicity and probably confers extended duration of protection.

15. Isolate related and designed attenuated in traditional method and vector based organisms to deliver foreign gene products in r-DNA. The improvement of hepatitis B vaccine may also induce faster and longer lasting protective immune responses in all vaccine recipients and in poor responders in particular R-DNA can be produced easily and can be inserted into multiple carriers. r-DNA vaccines include their purity in preparation , stability and safe use. RECOMBINANT DNA VS PLASMA DERVIED VACCINE

16. ANTIGEN ISOLATION The fragment of the gene is cleaved from the viral genome using RE enzyme EcoR1 This gene(with AUG)is joined to ADH promoter near the yeast alcohol dehydrogenase of plasmid vector pMA -56 This recombinant plasmid is introduced into the yeast cells

17. Yeast Human hepatitis B virus vaccine is prepared using antigen produced by recombinant technology in yeast (Saccharomyces cerevisiae). The highly purified antigen had the correct amino acid sequence and assumed the appropriate conformational structure to present the immunologic determinants (epitopes) that are needed to stimulate an appropriate immune response. Yeast-derived vaccine, is safe and is equally immunogenic and protective against hepatitis B as plasma-derived vaccine, as demonstrated in tests carried out in animals and in human beings. The yeast-derived vaccine produced by the Merck Sharp & Dohme Research Laboratories RECOMBIVAX HB was licensed for general use in the Federal Republic of Germany in May and in the United States of America on July 23, 1986. It represents the first licensed vaccine of any kind produced by recombinant technology, and establishes the precedent for new vaccines to be made using this methodology. Host used in hepatitis b vaccine

18. MAMMALIAN CELL YEAST has a number of disadvantages, including the fact that the antigen is internal and the yeast must be opened to release the recombinant product. Furthermore , the yeast is unable to provide the same post translational modifications, protein folding, macromolecular assembly, and glycosylation , as observed in infected human hepatocytes , properties which are important for inducing enhanced immunogenicity, and which are present in mammalian cells Available information suggests that Chinese hamster ovary (CHO) cell line and mouse-cell line-derived pre-S/S hepatitis B vaccines seem to be more immunogenic on the T-cell level (even with the use of alum as an adjuvant). Such vaccines were shown to generate T-cell help leading to higher seroconversion rates and anti-HBs titers at lower doses, as compared to yeast-derived SHBsAg The current safety record of mammalian cell-derived biological products, and especially CHO-derived antigens, is excellent, and a number of such products are already licensed. A ‘third-generation’ mammalian cell-derived vaccine was first developed at the Pasteur Institute in transfected CHO cells, expressing S and pre-S2 antigens

19. Expression OF HbsAg gene in yeast cells Specific activity can be increased many fold when cells are grown in shake flasks containing nonselective rather than selective medium. The addition of adenine, ammonium sulfate or glucose to the complex medium reduces the production of antigen in yeast cells. The addition of glucose to the culture medium can increase cell mass around 6-fold but can decrease the production of antigen. Therefore , A solution of yeast extract, soy peptone and glucose instead of glucose can be used to increase the antigen production in yeast cells.

20. Use of fermentors to scale up the process Relative abundance of HBsAg remains constant in both shake flasks and fermenters ; therefore, proportionally more HBsAg are produced when the fermentation process is scaled up. By doubling the concentrations of the components of Yeast growth medium, the cell mass can be increased. Since the relative abundance of antigen remains constant, the amount of HBsAg produced also be increased. A fed-batch fermentation process can be developed, in which the rate of glucose addition is increased and also a solution yeast extract, soy peptone and glucose instead of only glucose.

21. Isolation and Purification Purification selectively separates and retains the desired product at the highest purity per its pre-determined specification. (Remove unwanted compounds)• The most common method of vaccine production is based on an initial fermentation process followed by purification Methods followed are CENTRIFUGATION • FILTRATION • CHROMATOGRAPHY CENTRIFUGATION : Centrifugation is a process by which solid particles are sediment and separated from a liquid using centrifugal force as a driving force. Centrifugation is also used to remove dead cells, cell debris etc.• Example : Influenza vaccine, rabies vaccine ,Hepatitis B vaccine Centrifugation methods used for purification are – 1 . Differential Centrifugation 2 . Density gradient Centrifugation

22. Chromatography A group of physical separation techniques, which are characterized by the separation of mixtures due to differences in the distribution coefficient of sample components between two phases, one stationary and the other mobile phase . Example : Modified Vaccinia Ankara virus (Small pox vaccine ) Column Chromatography:-• Separates molecules by their chemical and physical differences . Most commonly used column chromatography are Ion exchange chromatography:- Separation on the basis of charge . Cell culture-derived inactivated whole virus vaccines Affinity chromatography :- Separation on the basis of specific binding sites on the protein.• Recombinant human glycoproteins• Cell culture-derived influenza virus particles .

23. Filtration Filtration is classified in two ways. 1 . DEAD END FILTRATION :-• all the flows are directed through the membrane with material building up on the surface of filter. (Flow perpendicular to membrane surface )• As these particles build up, flow through the filter is quickly reduced and finally it ceases completely. (Causes build up of filter cake on membrane ) 2 . TANGENTIAL FLOW FILTRATION (CROSS FLOW TECHNOLOGY) :-During CFF, culture fluid is re-circulated in tangential flow, parallel to the filter membrane. Build-up of viral particles on the membrane is minimized by there circulation of fluid over the surface, which also facilitates the concentration of particles present in the retained fluid. Mainly used in purifying inactivated Arboviral antigen. Ultrafiltration :-• A technique for separating dissolved molecules in solution on the basis of size rating the particles will be retained at the surface of the membrane.• During this process the desired proteins and their allied products are separated by their molecular weight, and the volume is reduced thereby increasing the purity considerably compared to the starting volume

24. FORMULATION OF VACCINE Active ingredients:- ANTIGENS ALUMINIUM :– in very small amount & it strengthens and lengthens the immune response to vaccine. YEAST PROTEINS from yeast:- ting quantity may remain present in vaccine. FORMALDEHYDE :- to inactivate/kill HBV used in vaccine. SODIUM/POTASSIUM SALTS:- acidity regulators .

25. ADJUVANTS — enhance vaccine immunogenicity Aluminium gels or salts (Alum) used in several licensed vaccines which include : Di phtheria-pertussis-tetanu s Di phtheria - tetanus ( DT) DT combined with Hepatitis B (HBV ) Haemophilus influenza B Inactivated polio virus Hepatitis A ( HAV) Streptococcus pneumonia vaccine Meningococcal vaccine Human papilloma virus (HPV)

26. CONCLUSION

27. THANK YOU JASPREET KAUR KRITIKA MANAV MAYANK MUSKAAN NIPUN NISHA RITIKA MUZAMIL NITIKA PAAVAN PIYUSH POOJA PRANJAL PARDIS PRIYESH RAMANDEEP