In a world grappling with infectious diseases and global health challenges, the presentation titled "Vaccine Development: From Concept to Early Clinical Testing" is a captivating and informative exploration of the intricate journey vaccines undergo before reaching the crucial stage of early clinical testing. This presentation delves into the remarkable and often arduous process of turning scientific concepts into potential life-saving vaccines, highlighting the vital role they play in safeguarding public health.

1. Reecha Sharma
(2019BS49M)
M.Sc Zoology (2019-2021)
Department of Zoology and Aquaculture, COBS&H
CCS Haryana Agricultural University, Hisar
email: reecha151@gmail.com

2. At a Glance
• One of the brightest chapters in the history of science is the impact
of vaccines on human longevity and health.
• Vaccines have a history that started late in the 18th century.
• From the late 19th century, vaccines
could be developed in the laboratory.
• However, in the 20th century, it became
possible to develop vaccines based on
immunologic markers.
• In the 21st century, molecular biology
permits vaccine development that was
not possible before.
Plotkin, 2014

3. Introduction : Immune system
The immune system evolved to protect multicellular organisms from
pathogens. Highly adaptable, it defends the body against invaders as
diverse as the tiny intracellular virus that causes polio and as large as
the giant parasitic kidney worm Dioctophyme renale. This diversity of
potential pathogens requires a range of recognition and destruction
mechanisms to match the multitude of invaders. To meet this need,
vertebrates have evolved a complicated and dynamic network of cells,
molecules, and pathways.
Polio virus under microscope The parasitic Kidney Worm
(Book : Kuby Immunology by Punt)

4. Natural immunity against a pathogen derives from the integrated
activation of the innate and adaptive immune systems.
Key characteristics and effectors cells of the innate and adaptive immune response:
Innate Immunity: First line of defense Adaptive Immunity: Second line of defense
• Triggered by damage or threat
(recognition of PAMPs)
• Activated by pathogen encounter
• Rapid response (hours) • Slower response (days or weeks)
• Usually no development of memory • Development of memory
• Pathogen destruction via phagocytosis,
killing and release of bioactive
mediators
• Antibody mediated and T-cell mediated
destruction
• Stereotypical response • Highly specific and adaptable
• Effector cells: Granulocytes , Mast cells,
Macrophages, Monocytes, Natural killer
cells, Dendritic cells
• Effector cells: CD4+ T-cells, CD8+ T-cells,
B-cells, Plasma cells
Cunningham et al , 2016

5. • Innate immunity arises after detection of specific pathogen-
associated molecular patterns (PAMPs) through a variety of pattern
recognition receptors (PRRs) and depending on the type of PAMP,
activate specialized Antigen Presenting Cells (APCs) e.g., dendritic
cells.
• Activation of innate immunity induces expansion of adaptive
immune cells targeted to the particular threat through antigen-
specific T-cell effector and antibody mechanisms. The
immunological memory derived from this antigen-specific response
persists and can react more rapidly upon subsequent infection .
• Immunization is the strategy of stimulating the host’s defense
against a specific pathogen to establish immunological memory and
thus protect against the consequences of infection.
Cunningham et al , 2016

6. Vaccination : Definition
• Vaccination is the administration of the vaccine to help the
immune system develop protection from disease.
• Vaccine is a biological preparation that provides active acquired
immunity to a particular infectious disease.
• A vaccine typically contains
an agent that resembles a
disease-causing microorganism
and is often made from weakened
(attenuated) or killed forms of the
microbe, its toxins, or genetically
engineered etc.
https://en.wikipedia.org/

7. The Birth of Vaccinology
• The genesis of vaccinology came through the evolution of attempts to
control smallpox. The observation that persons who had contracted smallpox
rarely developed a second case suggested the concept of immunity to
disease.
• The Chinese are generally credited with the development of variolation more
than a thousand years ago. This method of prevention spread westward into
Europe in the 17th century, although results were mixed and significant
complications often ensued.
• In 1796, Edward Jenner combined the practice of variolation with the
observation that milkmaids who had previously contracted cowpox rarely
contracted smallpox, and he performed the first immunization by inoculating
an 8-year-old boy with fluid from a cowpox pustule and later intentionally
infected the child with smallpox. As predicted, the child did not develop
smallpox. (McCullers, 2007)
• His assertion “that the cow-pox protects the human constitution from the
infection of smallpox” laid the foundation for modern vaccinology.
(Stern and Markel, 2005)

8. Timeline of Vaccine Development
Live Attenuated Killed whole
organisms
Purified proteins
and polysaccharide
Genetically engineered
18th Century
Smallpox(1796)
19th Century
Rabies (1885) Typhoid (1896)
Cholera (1896)
Plaque (1897)
Early 20th Century, 1st half
Tuberculosis (BCG) (1927) Pertussis (1926) Diphtheria (1923)
Yellow fever (1935) Influenza (1936) Tetanus toxoid(1926)
Rickettsia(1938)
Early 20th Century, 2nd half
Polio (Oral) (1963) Polio (injected)
(1995)
Anthrax secreted
protein (1970)
Hepatitis B surface
antigen Recomb.(1986)

9. Live attenuated Killed whole
organism
Purified protein and
Polysaccharide
Genetically
engineered
Measles (1963) Rabies (cell culture)
(1980)
Meningococcus poly-
saccharide (1974)
Lyme OspA (1998)
Mumps(1967) Japanese encephalitis
(1992)
Pneumococcus poly-
saccharide (1977)
Cholera (Recomb.
Toxin B) (1993)
Rubella (1969) Tick born encephalitis
(1981)
Haemophilus influenza
type B polysach.(1985)
Adenovirus(1980) Hepatitis A (1996) H. Influenza type B
Conjugate (1987)
Typhoid (1989) Cholera (1991) Typhoid polysach. (1994)
Varicella (1995) Meningococcal
conjugate (1999)
Acellular pertussis (1996)
Rotavirus (1999) Hepatitis B (1981)
Cholera (1994)
Cold adapted
influenza (1999)

10. Plotkin, 2014
Live attenuated Killed whole
organism
Purified protein and
Polysaccharide
Genetically
engineered
21st Century
Rotavirus (attenuated
and new
reassortment) (2006)
Japanese
encephalitis (2009)
Pneumococcal
conjugates
(heptavalent) (2000)
Human
papillomavirus
recomb. (4 valent)
(2006)
Zoster (2006) Cholera (WC only)
(2009)
Meningococcal conj-
ugates (3 valent) (2005)
Human P. Virus
(bivalent) (2009)
Pneumococcal conj-
ugates (13 valent)
(2010)
Meningococcal group
B proteins (2013)

11. Progress of polio elimination 1988 and 2014
Source : Centers for disease control and prevention (CDC)

12. How do vaccine work
World Health Organisation (WHO)

13. The goal of all vaccines is
to elicit an immune
response against an
antigen so that when the
individual is again
exposed to the antigen,
a much stronger
secondary immune
response.

14. Properties of an Ideal Vaccine
1) Effectiveness
• The vaccine would be effective against all current and future strains of the pathogen.
• Should give life long immunity.
• Must induce effective herd immunity.
• Must evoke protective levels of immunity rapidly.
2) Safety
• The vaccine should be 100% safe to the recipient.
• An ideal vaccine should not have any potential to disease or lead to any vaccine
associated diseases like allergic response, local inflammation, fever etc.
3) Availability : Readily cultured in bulk or accessible source of subunit and cheap.
4) Stability: Stable under extreme climatic conditions, preferably not requiring
refrigeration.
5) Dose : Only one dose required with easy administration.
6) Compatibility : Delivery of the vaccine simultaneously with other vaccines would
be possible. Many vaccines today are delivered simultaneously with other vaccines.
E.g. DTP and MMR.

16. Herd Immunity
When the vaccination of a portion of the population provides protection
to unprotected individuals is termed herd immunity.
Source : Centers for disease control and prevention (CDC)

17. Development of vaccine
Any new vaccine development requires integration of information concerning :
1. Pathogen life-cycle & epidemiology
2. Immune control & escape
3. Antigen selection & vaccine formulation
4. Vaccine preclinical & clinical testing

18. Pathogen Life-cycle and epidemiology
• Understanding the epidemiology of the disease is crucial to
identifying the target antigens.
• In order to identify antigens suitable for disease prevention, detailed
knowledge required are :
Biology and
Structure of
the pathogen
Interaction
with cellular
receptors
Disease
causing
mechanism

19. • Knowledge of the route of entry and subsequent replication sites of
the pathogen is also essential.
• This is because protection against pathogens entering via the
respiratory (influenza, pneumococcus), gastrointestinal (Salmonella)
or genital tracts (Herpes simplex virus [HSV] or human
immunodeficiency virus [HIV]), or entering the bloodstream by
injury/injection (hepatitis B/C) or mosquito bite (Malaria), may
require different vaccination strategies.
• For example, the immune response after natural malaria infection is
considered to be predominantly directed against the blood stage of the
pathogen but some vaccines have shown that it is possible to induce
effective immunity by targeting the pre-erythrocytic stage, e.g. during
sporozoite stage and the liver stage of the pathogen.
Cunningham et al , 2016

20. Life cycle stages of Plasmodium and vaccine candidates that target each stage.
Duffy and Gorres, 2020

21. Immune control & escape
Sometimes pathogens express a number of virulence determinants that
allow the pathogen to avoid immune defenses, facilitating infectivity
and transmission due to its considerable genetic diversity within its
species or virus type.
Immune evasion strategies :
1) Hiding from the immune system: Viruses such as Varicella
zoster (chickenpox) and Herpes viridae (herpes simplex viruses)
can hide from the immune system in neurons and non-neuronal
cells where they may persist for many years, before emerging in
pathogenic form when the host has a lowered resistance.
2) Interfering with the function of the immune system :
• Pathogen-encoded determinants bind to specific cellular receptors
allowing cell entry without alerting the immune response (viruses
and some bacteria) www.immunology.org

22. • Production of proteins, enzymes and micro-RNAs that inhibit host-
pathogen recognition mechanisms and innate immune effector
responses (influenza A)
• Production of structures such as polysaccharide capsules that inhibit
immune effectors such as complement. (Neisseria meningitidis)
• High rates of mutation that ensure that antibodies stimulated during
earlier infection remain ineffective (influenza, HIV, hepatitis C)
• Expression of toxins that cause tissue destruction and modulate the
immune response (pneumococcus)
Detailed knowledge of immune escape strategies used by individual
pathogens is important for developing effective vaccines against them.
Cunningham et al , 2016

24. Antigen selection & vaccine formulation
• There are many types of vaccines, categorized by the antigen used in
their preparation.
• In principle anything from whole organism to small subcellular
fragment can be used as antigen in vaccine.
Whole organism Vaccine
 Live but attenuated vaccines
 Inactivated (Killed) vaccine
Purified antigen vaccine or subunit vaccine
DNA vaccine
Recombinant vector vaccine

25. Live Attenuated Vaccine
• An attenuated vaccine contain a group of microbes that had been
weakened and decreased its virulent under laboratory conditions
(unfavorable conditions).
• The microorganisms lose their virulence and do not produce any type
of lesion in the animal, but continue to be able to replicate or multiply
sufficiently in order to be processed by the immune system .
(Saadh et al , 2017)
Plotkin, Vaccine Fact Book , 2013

26. (Book : Kuby Immunology by Punt)
Advantages
• Due to their capacity for transient growth,
these vaccine show prolonged
immunogenicity and eliminate the need for
repeated boosters.
• Infectious microbes can stimulate
generation of memory as well as humoral
immune response.
Disadvantages
• Major disadvantage of these vaccine is the
associated risk of reverting back to
virulent form.
• Since they are alive and their activity
depends on their viability, proper storage
is critical.

27. https://www.biodiem.com/

28. Example:
• Bacille Calmette-Guérin (BCG) is a vaccine against tuberculosis,
developed in 1921 by Albert Calmette and Camille Guérin, caused
by attenuation of the Mycobacterium bovis strain on a medium
containing increasing concentration of bile.
• The sabin polio vaccine is an example of attenuated vaccine,
consisting of three attenuated strain of poliovirus.
• Mumps vaccine consists of live attenuated strains of Paramyxvirus
parotitidis. In many world regions, it is used to routinely vaccinate
children, often a part f a combined measles, mumps and rubella
(MMR).
• Rabies vaccine, Yellow Fever vaccine, Vibrio cholera vaccine , Rota
virus vaccine.

29. Inactivated (killed) Vaccine
 Inactivated vaccines or killed vaccines is a vaccine consist of virus,
bacterial or other pathogens that have been grown in a specialized
culture and then completely killed by heat, radiation or chemicals so it
is no longer capable of replication in the host and to be effective must
contain much more antigen than live vaccines.
(Saadh et al , 2017)
Plotkin, Vaccine Fact Book , 2013

30. • Inactivated process is critically important to maintain the structure of
epitopes on surface antigens during inactivation.
• Excessive heat inactivation cause denaturation of protein, therefore
the epitope depend on structure of protein are altered or damaged.
• So the most successful methods to kill the pathogen depend on
chemicals such as Formaldehyde, phenol, and binary ethylenimine
(BEI).
• Excessive treatment can destroy immunogenicity whereas insufficient
treatment can leave infectious virus capable of causing disease
Example:
 The Salk inactivated polio vaccine (IPV) is produced by formaldehyde
treatment of the poliovirus.
 Hepatitis A

31. Subunit Vaccine (Purified Antigen Vaccine)
• A vaccine composed of a purified Antigenic determinant that is
separated from the virulent organism is called subunit vaccine.
• It consists of only those antigens that elicit protective immunity.
Subunit
Vaccine
Inactivated
exotoxin
Capsular
polysaccharide
Recombinant
antigen
World Health Organization (WHO)

32. Toxoid (Inactivated Exotoxin)
• Toxoid vaccines are made by purifying the bacterial exotoxin.
• Toxicity of purified exotoxins is suppressed or inactivated either by
heat or with formaldehyde (while maintaining immunogenicity) to
form toxoids. Vaccination with toxoids induces anti-toxoid
antibodies that are able to bind with the toxin and neutralize its
deleterious effects. (Yadav et al, 2014).
• Example : DPT
Vaccine Research Catalog, 2016

33. Capsulated Polysaccharide Vaccine
• Bacteria can synthesize hundreds of chemically and
immunologically different polysaccharides.
• Vaccines composed of purified polysaccharides against
meningococcus and pneumococcus were developed .
• Unfortunately, those vaccines, while partially immunogenic in
adults, were completely unable to induce an antibody response in
infants and children, the population for whom the vaccines were
mostly needed.
• Later the problem was solved and reported that the bacterial CPSs
become very immunogenic when covalently linked to a carrier
protein and thus started working on a conjugate vaccine which
worked beautifully in infants and children.
(Rappuoli et al , 2019)

34. Pure polysaccharide V/S Conjugate vaccine
Rappuoli et al , 2019

35. Conjugate Vaccine
• A conjugate vaccine is created by covalently attaching a poor antigen
(polysaccharide) to a strong antigen (protein/ toxoid) thereby eliciting
a stronger immunological response to the poor antigen.
• Example:
 Haemophilus influenza type B (Hib) Vaccine
 Pneumococcal
Vaccine
 Meningococcal
Vaccine

36. Recombinant Subunit Vaccine
• The use of recombinant DNA technology has made the
development of subunit vaccine more efficient.
• Recombinant subunit vaccines can be delivered as purified
recombinant proteins, as proteins delivered using live non-
pathogenic vectors (bacterial or viral) or as nucleic acid molecules
encoding the antigen, thereby making the production safer and
generally more efficient.
• The first Recombinant subunit vaccine approved for human use is
the Hepatitis B vaccine.
(Andersson, 2000)

38. DNA Vaccine
• The DNA vaccines are simple rings of DNA containing a gene
encoding an antigen, and a promoter/terminator to make the gene
express in mammalian cells.
• They are a promising new approach for generating all types of
desired immunity: cytotoxic T lymphocytes (CTL), T helper cells
and antibodies.
Liu, 2003

40. Liu, 2003

41. Advantages of DNA Vaccines
The main advantage of DNA vaccines is their ability to
stimulate both the humoral and cellular arms of the
adaptive immune system.
Versatility: In addition to the prevention of
infectious diseases, DNA vaccines may also be used
to treat malignancies and autoimmune or genetic
disorders.
Due to the ability to genetically modify the antigen
encoded by DNA vaccines, the vaccine provides a
means to generate broadly neutralizing antibodies
against pathogens such as HIV and the influenza
virus.
Flingai et al , 2013

42. Recombinant Vector Vaccine
• In this, genes that encode antigens isolated from a pathogen can be
inserted into non-virulent viruses or bacteria.
• Such recombinant micro-organisms serve as vectors, replicating
within the host and expressing the gene product of the pathogen-
encoded antigenic proteins.
• A no. of organisms have been used for vector vaccines, including
vaccinia virus, the canary pox virus, attenuated polio virus,
adenovirus and others. (Book : Kuby Immunology by Punt)

44. Formulation of Vaccine
Vaccines include a variety of ingredients including antigens, stabilizers,
adjuvants, antibiotics, surfactants, diluent, residual and preservatives.

45. Antigen
 All vaccines contain an active component (the antigen) which
generates an immune response, or the blueprint for making the active
component.
Stabilizers
 Stabilizers are used to help the vaccine maintain its effectiveness
during the storage.
 Instability can cause loss of antigenicity and decreased infectivity of
LAV.
 Factors affecting stability are temperature and acidity or alkalinity of
the vaccine (pH).
 Stabilizing agents include MgCl2 (for OPV), MgSO4 (for measles),
lactose-sorbitol and sorbitol-gelatine.

46. Adjuvants
Added to vaccines to stimulate the production of
antibodies against the vaccine to make it more
effective.
Chemically, adjuvants are a highly heterogeneous group of
compounds with only one thing in common: their ability to
enhance the immune response.
Example : Aluminium salt (like aluminium phosphate,
aluminium hydroxide or potassium aluminium sulphate),
CpG ( Two nucleic acids, Cytosine(C) and Guanine (G)
found in the DNA) are linked to form an adjuvant that is
contained in a hepatitis B vaccine.

47. Lambert et al , 2005

48. 17 February 2020

49. Antibiotics
 Antibiotics (in trace amounts) are used during the manufacturing
phase to prevent bacterial contamination of the tissue culture cells in
which the viruses are grown.
 Usually only trace amounts appear in vaccines, for example, MMR
vaccine and IPV each contain less than 25 micrograms of neomycin
per dose (less than 0.000025 g).
 Example: Neomycin, streptomycin, polymyxin B, chlortetracyline
and amphotericin B.
WHO

50. Preservatives
 Preservatives are added to multidose vaccines to prevent bacterial
and fungal growth. They include a variety of substances.
 Example:
 Thiomersal (ethyl mercury-containing compound)
 Formaldehyde(Used to inactivate viruses (e.g. IPV) and to detoxify
bacterial toxins, such as the toxins used to make diphtheria and
tetanus vaccines).
 Phenol derivatives.
Surfactants
 Surfactants keep all the ingredients in the vaccine blended together.
They prevent settling and clumping of elements that are in the liquid
form of the vaccine.

51. Residuals
 Residuals are tiny amounts of various substances used during
manufacturing or production of vaccines that are not active
ingredients in the completed vaccine.
 Substances will vary depending on the manufacturing process used
and may include egg proteins, yeast or antibiotics. Residual traces of
these substances which may be present in a vaccine are in such small
quantities that they need to be measured as parts per million or parts
per billion.
Diluent
 A diluent is a liquid used to dilute a vaccine to the correct
concentration immediately prior to use. The most commonly used
diluent is sterile water.
WHO

52. Route of Administration
World Health Organization (WHO)

53. Source : Centers for disease control and prevention (CDC)

54. Vaccine Preclinical & Clinical Testing

55. Clinical Development

56. Regulatory Review and Approval
 This step can only come after detailed applications and rigorous
demonstration of Good Manufacturing Practices.
 Regulatory review and approval processes then allow the
manufacturer to receive FDA (Food and Drug Administration)
licensing.
 The process includes :
 An Investigational New Drug application,
 Pre-licensure vaccine clinical trials,
 A BLA (Biologics License Application),
 Inspection of the manufacturing facility,
 Presentation of findings and usability testing of product labeling.

57. Manufacturing
 During this part of the process, vaccines are made.The production
takes between six and 36 months. Several hundreds of quality tests
will take place during that span.

59. Quality Control
 Using information from the public and healthcare providers, quality
control continues through different agencies/methods long after a
vaccine is licensed, manufactured and distributed.
 As soon as public vaccine use starts, vaccine performance is
constantly checked. Multiple systems and organizations monitor and
test the vaccine.
Centers for disease control and prevention (CDC)

60. Vaccine Development
in
Global Pandemic

61. How do vaccine work against Covid19 ?
 Scientist and researchers worldwide race to develop a vaccine in the fight against
global pandemic (COVID-19) where main focus is to the so-called spike protein,
which is present in the COVID virus.
 The spike protein interacts with ACE2
proteins on human cells, facilitating
infection of the cells, encouraging the
virus to replicate and cause disease.
 Several vaccines in development are
aimed at exposing the body to spike
protein and having the immune system
recognize it as an antigen, or foreign.
 The immune system then develops a
response with specialized white blood cells that are aimed at either destroying the
virus or producing antibodies that will block infection by the virus when exposed to the
real pathogen.

62. Host Immune Response against SARS-CoV2-infection
Prompetchara et al, 2020

64. The global COVID-19 vaccine landscape
World Health Organization (WHO)

67. Phase 3 Covid Vaccine
Sputnik V
mRNA-1273
Ad5
Covaxin

68. Conclusion
• Vaccination as a means of preventing infectious disease has had
arguably the greatest impact on human health of any medical
intervention.
• Vaccine development is a complex multidisciplinary activity,
combining understanding of host-pathogen interactions at the
molecular level, with clinical science, population-level
epidemiology and the biomechanical requirements of production
• Increased understanding of the molecular nature of immune
responses and advances in the technologies of gene sequencing
and molecular biology have resulted in new approaches to vaccine
development.
• The ultimate goal is an affordable vaccine that generates strong
and lasting immunity with the fewest possible side effects,
implemented without the need for expensive cold chains.

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71. SYMBOL OF TRUST