The document discusses recombinant DNA technology for producing hepatitis B vaccines. It begins by introducing the different types of hepatitis viruses and diseases. It then describes the structure and life cycle of the hepatitis B virus. The key aspects of producing recombinant hepatitis B vaccines are using the viral surface antigen gene isolated from the hepatitis B virus genome and expressing it in host cells like yeast or mammalian cells. The vaccines produced this way have been found to be safe and effective alternatives to plasma-derived vaccines.

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

3. 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.

4. ● 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 :-

5. ● Acute hepatitis (self-limited liver injury of
less than 6 months
● Chronic 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.

6. 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.

7. 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.

8. 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 )

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

10. 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.

11. 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.

12. 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

13. 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

14. 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)

15. 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
• The 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.

16. • 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

18. 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

19. 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

20. 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

21. 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.

22. 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.

23. 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

24. 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.

25. 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

27. 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.

28. ADJUVANTS —enhance vaccine immunogenicity
Aluminium gels or salts (Alum) used in several licensed
vaccines which include:
• Diphtheria-pertussis-tetanus
• Diphtheria - 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)

29. CONCLUSION

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