vaccine development.pptx

CELL CULTURE BASED VACCINE
General introduction, Vaccines for Malaria andAIDS
January 29, 2020
1

Introduction
⚫ Cell culture based vaccine??
⚫ Cell cultures involve growing cells in a culture dish, often with a
supportive growth medium. A primary cell culture consists of cells taken
directly from living tissue, and may contain multiple types of cells such as
fibroblasts, epithelial, and endothelial cells.
⚫ In the United States, 10 different vaccines for chicken pox, hepatitis A,
polio, rabies, and rubella are cultured on aborted tissue from two fetal
cell lines known as WI-38 and MRC-5. These vaccines are chicken pox,
hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella,
mumps/rubella, and MMR II (measles/mumps/rubella).
January 29, 2020
2

History
⚫PCCs of monkey kidney cells have been used for the
production of inactivated and oral poliomyelitis vaccines
since the 1950s.
⚫In 1954, an experimental adenovirus vaccine was being
developed, and human tumour cells (HeLa) were
rejected as the cell substrate in favour of ”normal” cells.
⚫The first requirements for cell substrates were published
by WHO in 1959 for the production of inactivated
poliomyelitis vaccine in PCCs derived from the kidneys
of clinically healthy monkeys.
January 29, 2020
3

History
⚫In the 1960s, human diploid cells (HDCs) were
developed and proposed as an alternative to primary
monkey kidney cell cultures for polio virus vaccine
production as well as for other viral vaccines.
⚫WHO Requirements for Continuous Cell Lines used for
Biologicals Production were published in 1987.
⚫During the 1990s, and on into the 2000s, a variety of CCLs
were explored as cell substrates for biological products.
January 29, 2020
4

Types of animal cell substrates
1. Primary Cell Cultures (PCCs)
o Major successes in the control of viral diseases, such as
poliomyelitis, measles, mumps and rubella, were made
possible through the wide use of vaccines prepared in
PCCs, including those from chicken embryos and the
kidneys of monkeys, dogs, rabbits and hamsters.
Advantages:
(a) Easy to prepare using simple media and bovine serum;
(b) Broad sensitivity to various viruses, some of which are cytopathic.
Disadvantages:
(a) Contamination by infectious agents is a higher risk than
with DCLs and CCLs;
(b) They cannot be tested as extensively as DCLs or CCLs.
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Advantages:
Disadvantages:
with DCLs and CCLs;
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January 29, 2020 7
Advantages:
Disadvantages:
with DCLs and CCLs;

2. Diploid Cell Lines (DCLs)
The practicality of using human DCLs for the
production of viral vaccines was demonstrated in the
1960s. DCLs of human (e.g., WI-38, MRC-5) and
monkey ( i.e., FRhL2 ) origin.
Advantages
(a) They can be well characterized and standardized;
(c) Unlike the CCLs and SCLs , DCLs are not tumourigenic and therefore do not
raise the potential safety issues associated with CCLs and SCLs.
Disadvantage
(a) In general, they have more fastidious nutritional requirements than other cell
substrates;
(b) They may be difficult to adapt to serum-free growth;
(c) They are more difficult than CCLs to transfect and engineer, and require
immortalization before they can be engineered;
January 29, 2020 8

The practicality of using human DCLs for the production of viral
vaccines was demonstrated in the 1960s. DCLs of human (e.g.,
WI-38, MRC-5) and monkey ( i.e., FRhL2 ) origin.
Advantages
(c) Unlike the CCLs and SCLs , DCLs are not tumourigenic and
therefore do not raise the potential safety issues associated
with CCLs and SCLs.
Disadvantage
substrates;
January 29, 2020 9

January 29, 2020 10
The practicality of using human DCLs for the production of viral
vaccines was demonstrated in the 1960s. DCLs of human (e.g.,
WI-38, MRC-5) and monkey ( i.e., FRhL2 ) origin.
Advantages
(c) Unlike the CCLs and SCLs , DCLs are not tumourigenic and therefore do not
raise the potential safety issues associated with CCLs and SCLs.
Disadvantage
substrates;

January 29, 2020 11
3.Continuous cell lines (CCLs)
CCLs have the potential for an apparently indefinite in vitro life span
and have been derived by the following methods:
a) Serial sub cultivation of a PCC of a human or animal tumor (e.g., HeLa
cells);
(b) Transformation of a normal cell having a finite life span with an
oncogenic virus or viral sequence (e.g., B lymphocytes transformed by
EBV or transfected with viral sequences such as in PER.C6);
(c) Serial sub cultivation of a primary-cell population derived from normal
tissue that generates a dominant cell population having an apparently
indefinite life span, often described as spontaneous transformation
(e.g., Vero, BHK-21, CHO, MDCK, Hi5);
(d) Fusion between a myeloma cell and an antibody-producing B
lymphocyte to produce a Hybridoma cell line; or

Advantages:
(a) Characterized extensively and their culture conditions
standardized;
(b) Grow more easily than DCLs using standard media,
(d) Most can be adapted to grow in serum-free medium;
(e)Can be grown on microcarriers for large-scale production
in bioreactors;
(f) some can be adapted to grow in suspension cultures for
large-scale production in bioreactors.
Disadvantages
(a) CCLs may express endogenous viruses, and some are tumourigenic in
immunosuppressed animal models;
(b) Theoretical risks identified by the 1986 Study Group (e.g., nucleic acids,
transforming proteins, and viruses) need to be taken into account.
January 29, 2020
12

Advantages:
(a) Characterized extensively and their culture conditions standardized;
(b) Grow more easily than DCLs using standard media,
(d) Most can be adapted to grow in serum-free medium;
(e) Can be grown on microcarriers for large-scale production in bioreactors;
(f) some can be adapted to grow in suspension cultures for large-scale
production in bioreactors.
Disadvantages
(a)CCLs may express endogenous viruses, and some
are tumourigenic in immuno suppressed animal
models;
(b)Theoretical risks identified by the 1986 Study
Group (e.g., nucleic acids, transforming proteins,
and viruses) need to be taken into account.
January 29, 2020
13

GENERAL STEPS OF VACCINE PRODUCTION FROJ
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14

VACCINES FROM CELL CULTURE
15
 MMR Vaccine
 Polio vaccine
 Chickenpox (Varicella) vaccine
 Rabies vaccine
 Recombinant protein vaccines
 Influenza vaccine
 Dendritic Cell-Based Vaccination in Solid
Cancer
January 29, 2020

MMR Vaccine
Disease Immunized Virus Strain Propagation cell Growth Medium
Measles
Enders' attenuated
Edmonston strain
chick embryo cell
culture
Medium 199
Mumps
Jeryl Lynn(B level)
strain
chick embryo cell
culture
Medium 199
Rubella
Wistar RA 27/3 strain
of live attenuated
rubella virus
WI-38 human diploid
lung fibroblasts
MEM (solution
containing buffered
salts, fetal bovine
serum, human serum
albumin and
neomycin, etc.)
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16

Polio , Chickenpox and Rabies vaccine
Disease Immunized Virus Strain Propagation cell Growth Medium
Poliomyelitis or
polio
Type 1 (Mahoney),
Type 2 (MEF-1),
Type 3 (Saukett)
vero cells, a
continuous line of
monkey kidney
cells
Eagle MEM
modified medium,
M-199
Chickenpox Oka/Merck strain
(varicella virus)
Human diploid
cell cultures
(WI-38, MRC-5)
MEM
Rabies
Attenuated
Pitman-Moore
L503 strain
Attenuated Wistar
strain
Human diploid
cell
chicken embryo
Vero cell
Eagle MEM
modified medium,
M-199
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17

iNfluenza vaccine
January 29, 2020 18

January 29, 2020 22
AIDS vaccine development:
Perspectives, challenges & hopes

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Introduction
• In June 1981, the Center for Disease Control (CDC) in the United States
reported the first clinical evidence of a disease that would become known
as Acquired Immunodeficiency Syndrome (AIDS).
• 23 years later, the AIDS epidemic has spread all over the world. Since the
beginning of the epidemic, 63 million people have been infected with HIV.
• Globally, over 38 million people are today living with HIV infection, with 5
million new infections acquired annually, and 14,000 daily.
• Over 95 % of new HIV infections occur in developing countries, mainly in
Sub-Saharan Africa and South-East Asia.
• There is an urgent need to explore all possible approaches to control the
epidemic, in particular, preventive measures such as health education and
treatment of sexually transmitted diseases, preventive vaccines and
topical microbicides.

January 29, 2020 24
Introduction
• If considerable progress has been made in treatment with antiretroviral
drugs and their largescale implementation, the need for an AIDS vaccine
has spurred an unprecedented effort of research and development
worldwide, especially in South-East Asia .
• Preventive vaccines represent our only long-term hope to stop the
epidemic.
• Since the first report of HIV infection among sex workers in Chennai in
1986. it is estimated that over 5.1 million people were infected with HIV in
India by the end of 2003.
• The HIV epidemic has been spreading from high risk to low risk
populations and from urban to rural areas during the last 12-13 year.

January 29, 2020 25
https://www.avert.org/professionals/hiv-around-world/asia-pacific/india,
dated March 28,2018, 9.00 AM

January 29, 2020 26
Scientific obstacles to the development of
AIDS vaccines
• Antigenic diversity and hyper variability of the virus
• Transmission of disease by mucosal route
• Transmission of the virus by infected cells
• Resistance of wild type virus to seroneutralization
• Integration of the virus genome into the host cell
chromosomes
• Latency of the virus in resting memory T-cells
• Rapid emergence of viral escape mutants in the host
• Downregulation of major histocompatibility (MHC) class I
antigens

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Programmatic difficulties to the development of AIDS
vaccines
• Insufficient political leadership
• Insufficient funding's allocated to AIDS vaccines
• Lack or insufficient coordinated approach
• Regulatory authorities in developing countries
• Slow approval process
• Standardization of assays and reagents
• Length of clinical trials, especially of efficacy trials

HIV VACCINES
• Various approaches are being explored for the generation of
HIV vaccines, and the U.S. has committed itself to developing
such a vaccine by the year 2007. Since 1987, more than 40
different preventive
• HIV vaccines have been studied in clinical trials worldwide.
These have a variety of different strategies,
• Among the current approaches to HIV vaccine development, a number of
strategies merit special comment. These include the following:
January 29, 2020 28

January 29, 2020 30
HIV VACCINES
(1) Recombinant gp120
This is probably the most well studied candidate HIV-1 vaccine, but one
which fails to generate measurable CTL responses.
VaxGen, a San Francisco-based company, initiated the first Phase 3
efficacy trial of an AIDS vaccine in 1998 using its gp120 subunit vaccine
known as AIDSVAX.
The vaccine is safe, and it elicits a strong serologic response, with
measurable levels of homologous virus-neutralizing antibodies 5,000
volunteers will be enrolled in this Phase 3 study . A second Phase 3 trial
with AIDSVAX was initiated in Thailand in 1999. It is expected that these
efficacy trials will provide a definitive test for the hypothesis that gp120
subunit vaccines can elicit protective serologic immune responses against
HIV-1.
(2) Nucleic acid vaccines
Nucleic acid vaccine strategies have, to date, taken the form of DNA
expression plasmids encoding HIV- 1 gene products, which are usually
injected either intradermal or intramuscularly, using a Gene Gun or similar
device. Animal studies have shown that DNA vaccines can elicit CTL
responses and neutralizing antibodies against HIV and SIV antigens

HIV VACCINES
(3) Multivalent vaccines
A major concern with HIV-1 vaccination efforts is the extreme genetic and
antigenic diversity among HIV-1 strains, and among human populations . In an
effort to address these issues, some investigators are in the process of
developing multivalent or multimeric HIV-1 vaccine strategies.
Approaches include the use of multiple DNA or vaccinia virus (VV) vectored
gp120 immunogens , as well as the development of multiepitope “universal”
CTL immunogens capable of class-I restricted presentation by 90% of more of
the human population .
(4) Live-vectored vaccines
These include live-attenuated bacterial vectors, such as Bacille Calmette-Guerin
(BCG) and Salmonella . These vectors are particularly intriguing since they are
safe and can establish infection via a mucosal route .
Thus, they may elicit strong mucosal immune responses; testing of a
recombinant Salmonella-HIV gp120 candidate vaccine is presently in a phase I
trial (AVEG 029). New virus vector systems include improved poxvirus vectors
such as canarypox vectors and modified vaccinia Ankara (MVA), as well as other
vector systems, such as herpesviruses and Venezuelan equine encephalitis virus
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January 29, 2020 32
HIV VACCINES
(5) Combination approaches
The combination vaccine approaches elicit the most potent immune responses
in nonhuman primates and in humans.
As a result, several Phase I/II clinical trials of such approaches are now
underway. These include Phase I/II trials of vCP205, followed by (or
simultaneously with) a gp120 subunit boost, a p24 subunit boost or GMCSF.
Other combinations include a vaccinia virus-prime plus protein (gp120)-boost
strategy, a salmonella recombinant-prime plus protein boost strategy and a
DNA-prime plus protein or canarypox-boost.
(6) Plant based vaccines
Genetic engineering of plants has made it possible to explore the use of plant-
based immunogens. This approach has resulted in the production of
measurable immune responses to bacterial and viral antigens (including HIV-1)
in both experimental animals, and in humans . It is possible that plant-derived
products, including orally-delivered edible vaccines, may have future potential
in the context of AIDS vaccine development.

January 29, 2020 33
HIV VACCINES
(7) Live-attenuated vaccines
Initial studies with strains of simian immunodeficiency viruses (SIV) showed
that live-attenuated viruses can elicit immune responses that result in
protection from challenge with infectious SIV .
Subsequent findings with triply deleted SIV mutants have confirmed this ,
although it has also become apparent that
(1) there is variable level of vaccine protection by live attenuated SIV against
heterologous challenge ,and
(2) that the degree of immune protection by live attenuated SIV is inversely
correlated with the extent of viral attenuation
(3) The notion that live-attenuated vaccines for HIV may possess a significant
risk for causing AIDS is further.
(4) Nevertheless, the live-attenuated vaccine approach is a powerful and
potentially low-cost strategy that is too important to overlook within the
portfolio of AIDS vaccine approaches.
(5) Further explore the safety and immunogenicity of this approach, using
experimental animal models (Rhesus macaques)

4
ry 29, 2020 3
• Clinical trials
• First HIV vaccine trial to show a positive protective signal were
released in 2009. The trial, termed RV 144, was performed in
Thailand.
• It used a combination of two vaccines in a heterologous prime-
boost paradigm, i.e. one vaccine given in four doses was then
"boosted" by two doses containing both vaccines.
• Analysis of the trial showed that the group receiving the vaccine
had an infection rate 31.2% lower than the group that received
the placebo.
• Several trials are planned incorporating lessons from RV 144. If
the required efficacy can be shown in any of the trials, an
HIV/AIDS vaccine could become available from 2020.
The Army-led Thai HIV vaccine efficacy trial, known as RV144, tested the
“prime-boost” combination of two vaccines: ALVAC® HIV vaccine (the prime)
and AIDSVAX® B/E vaccine (the boost).
Janua

January 29, 2020 35
Types of Vaccine Gene Modes Study/Mode Remarks
Recombinant gp120 HIV-1 vaccine Fails to CTL responses
HIV-1 vaccine AIDSVAX (VaxGen, San Francisco)
1998
Phase 3 study Stong serological response ,
Nucleic acid Vaccine HIV -1 gene product Used IM, by gene gun Elicit CTL
Multivalent Vaccine HIV-1 Multiple DNA or Vaccinia virus (VV)
gp120
Elicit CTL
Live –vectored Vaccines HIV-1 BCG and Salmonella (AVEG029) Orally delivered edible
vaccines
Elicit CTL
Combination approaches gp120 subunit boost, a p24 subunit
boost,
Other combinations include a vaccinia
virus-prime plus protein,
DNA-prime plus protein or canarypox-
boost
Phase I/II clinical, Elicit CTL
Plant based vaccines HIV-1 bacterial and viral antigens orally-delivered edible
vaccines
Elicit CTL
Live-attenuated vaccines HIV-1 Simian immunodeficiency viruses
(SIV)
Elicit CTL

January 29, 2020 37
Current Milestones towards
Development of a Fully Deployable
Anti-Malaria Vaccine-Future Hope
for Malaria-Free World

January 29, 2020 38
Introduction
• Malaria ranks high among the most devastating parasitic diseases afflicting
humankind. The disease is caused by parasites of the genus Plasmodium ,
and is transmitted by female Anopheles mosquitoes .
• Plasmodium falciparum is the most virulent of the five known human
malaria parasites accounting for 90% of malaria-related deaths globally.
• The parasite is endemic within Sub-Saharan region of Africa On the other
hand, infections occurring outside this region are mainly attributable to P.
vivax which is less deadly compared P. falciparum.
• Interestingly, P. vivax remains dormant in the human liver, a unique feature
in its biology that accounts for its challenging elimination; which hampers
its elimination in endemic countries.
• Thus, malaria continues to exert robust health toll and economic burden
globally, more so in resource-constrained settings.

January 29, 2020 39
Introduction
• Standard immunization based on plasmodium whole-organism and
radiation-attenuated sporozoite (RAS), was assessed in mice, primates, and
humans over three decades ago,
• But became untenable as it required either several bites of irradiated
mosquitoes or intravenous inoculation of sporozoites, both of which were
considered impractical for mass vaccination .
• Likewise, research on Subunit protein or DNA- based vaccines has not
yielded desirable results with respect to malaria vaccine efficacy trials.
Consequently, this has triggered scaling up of P. falciparum RAS in order
optimize mass vaccination using this attenuated whole-organism approach.
• For instance, P. bergei genetically attenuated parasites (Pb GAP) and P. yoelii
GAP are observed to confer similar protection in experimental mice models.
• Ultimately, among the biggest drawbacks of developing effective malaria
vaccine would be the hardship of differentiating between the diversity
associated with immune escape as well as cross protection.

January 29, 2020 41
Introduction
• Breakthrough strategies are dependent on the identification of
immunologically relevant diversity through population genetics and
structural studies that identify functional polymorphisms.
• In addition, epidemiological surveys control the polymorphisms to be
considered when developing a multivalent malaria vaccine for multiple
strains.
• Overall, although considerable strides have been made based on different
approaches to come up with an effective malaria vaccine, quest for the
valuable tool remain unfulfilled
• Life cycle of Malaria.

Lifecycle of Malaria
Malaria is caused
by a unicellular
of the
eukaryotic
parasite
genus
plasmodium; 5
species including
P
. falciparum, P
.
vivax, P. ovale, P.
knowlesi
malariae
infectious
and P
.
are
to
humankind.
Parasitic forms in
sporozoite stage
are transmitted to
host
a bite
human
through
from a female
anopheles
mosquito
In extreme cases where the infected red blood
cells are sequestered in the brain, cerebral
malaria may occur with convulsions, followed
by coma, and eventual death
January 29, 2020 42

Current Advances towards Malaria Vaccinen Development
• The pre-erythrocytic vaccines including RTS S usually target the infective
sporozoite stage to prevent infection. In addition, they also focus on antigens
expressed by parasites at liver stage, by hindering release of merozoites into
the bloodstream which are responsible for symptomatic malaria.
• The major challenge associated with antigen targeting is that antigenic dose
released during natural infection by the vector is quite low to elicit an
effective immune response.
• Blood stage vaccines: form majority of candidate vaccines and as the name
denotes they control infection at the blood stage sub-level. They target
merozoite antigens to prevent red blood cell invasion, thereby reducing the
density and prevalence of parasites in the infected host. More novel
approaches targeting major surface proteins expressed on P. falciparum
infected red blood cell (erythrocyte membrane protein 1, PfEMP1) are
underway. However, the PfEMP1 mediates cyto-adherence to host cells and is
associated with severe malaria.
• Transmission blocking vaccines
Subunit vaccines that principally target gametocyte or oocyst antigens
expressed in the life stages of mosquito host . Such vaccines are not directly
involved in combating clinical disease but contribute enormously towards
Januaery
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a0tion efforts by hindering transmission. 45

• RTS,S. RTS,S (developed by PATH Malaria
Vaccine Initiative (MVI) and GlaxoSmithKline
(GSK) with support from the Bill and Melinda
Gates Foundation) is the most recently
developed recombinant vaccine
January 29, 2020 46

25/04/2017 on
January 29, 2020 49

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