Types of Vaccines

1
Purpose of Vaccination

2
 Vaccine Efficacy

• Diphtheria >87% (industrialized countries)

• Tetanus 3 doses >95%, 2 doses >80%
• Pertussis 80% (wide variation)
• Oral Polio >90% (lower in tropical countries)
• Measles >90% (after 12 mths) 85% (at 9 months)
• Hepatitis B 75 – 95%
• Hib 90%

3
 Normal Bacterial Flora

Staphylococcus epidermidis
Normal colon
Bacterial colonization of the body
per gm or cm2

Bifidobacterium bifidum
Bacterial colonization
4
E.coli
 Role of Bacterial Toxins in disease

DISEASE
TOXIN PRODUCED
(BACTERIA)
Diphtheria
Diphtheria toxin
Corynebacterium diphtheriae

Tetanus
Tetanus toxin
Clostridium tetani

Whooping cough Adenylate cyclase toxin

Bordetella pertussis
Pertussis toxin
Cholera
Cholera enterotoxin
Vibrio cholerae

5
 Invasion of Cells

Shigella bacterium invading HELA cell

6
 Basic concept of vaccines
• Deliver to the body some part or all of the disease
organism that IMITATES the pathogen but is not
pathogenic.
– Induce protective immune response.
LPS Polysaccharide

capsular Intracellular proteins

Entire organism Surface proteins

• live (attenuated)
• killed
Toxins

7
 New developments in vaccines

• Identifying the protective antigen

– Common antigens
– Poorly expressed antigens
• Expression of antigen
– Production system
• Production of protective antigens in yeast etc.
– Vector
• Live attenuated bacteria
• Live attenuated viruses

8
 New developments in vaccines

• Antigen presentation
• Virus like particles
• Adjuvants
• Slow release vehicles
• Conjugation
• Formulation
• Multivalent vaccines
• Addition of muco-adhesives for oral delivery
• Addition of preservatives and stabilizers (lyophilization)

9
 Vaccine manufacture
Antigen Production

• Eggs
– Influenza

• Bacterial / Yeast fermentation

– Whole organism (e.g. Cholera)
– Subunit vaccines (e.g. Capsular
polysaccharide, Tetanus and Diphtheria
toxoid)
– Genetically engineered proteins (e.g.
Hepatitis B and HPV vaccines)

• Cell culture
– Viral vaccines either whole virus or subunit
– Genetically engineered proteins
10
 Vaccine Processing
• Antigen concentration.
• Removal of unwanted foreign components.
– Host proteins
– Host DNA
– Adventitious agents
• Removal of unnecessary Bacterial or Viral
components.
– LPS
– Unwanted proteins (not involved in immunity)
– Unwanted nucleic acid
• Change of diluting solution.
• Addition of other components.
– Adjuvants, stabilizers, preservatives etc 11
 Growth
of
Virus, Yeast
Clarification
Concentratio
n + Purification
or Bacteria

Eggs Depth filter

Crossflow
filtration

Tools available to develop a process Chromatography

Ion exchange

Bacterial / Yeast Crossflow Hydrophobic

filtration Affinity
Fermentation
Ultracentrifugation Size exclusion

Centrifuge Chemical
Cell culture Formaldehyde
precipitation

Inactivation
βPL
12
 Antigen Fill and
presentation Formulation
Finish

DT CONHNHCO(CH2)4CONHNHOC- Vi-
CP
CONJUGATES S
Combining antigen(s)
Combining with
adjuvant
Stabilizers
VIRUS LIKE PARTICLES ISCOMS Preservatives
Cryo-protectants

VIROSOMES
ADJUVANTS 13
Types of Vaccines

14
Types of Vaccines
• Inactivated toxins
• Inactivated whole bacteria or viruses
• Live attenuated bacteria or viruses
• Subunit vaccines
• Genetically engineered proteins
• Polysaccharide vaccines
• Conjugated vaccines
• Recombinant DNA modified organisms
• DNA vaccines

15
 Inactivated Toxins

• Exotoxin
– Gram negative and gram
positive bacteria.
– Heat Labile.
– Protein.
– Secreted by the bacteria.
• Endotoxin
– Gram negative bacteria.
– Heat stable.
– Lipopolysaccharide.
– Firmly bound to the
bacteria outer membrane.

16
 Toxoid
• Not a vaccine against the organism.
• Vaccine against pathogenic exotoxin.
• Tetanus, diphtheria, (pertussis?), anthrax?
– Purify toxin then chemically inactivate (toxoid)
• Risk of incomplete inactivation.
• TT, DT.
– Genetically modify toxin so non-toxic
• CRM (diphtheria), mLT (cholera, ETEC)

17
 Inactivated Toxins

Tetanus toxin blocks Diphtheria toxin inhibits

neurotransmission from nerve protein synthesis resulting in
endings inducing muscle cell death
spasm and paralysis
18
Tetanus

19
 Tetanus

• Clostridium tetani
• Gram positive rod
• Not transmitted from person to person
• Transmission via contaminated wounds
• Found in soil and animal feces
• Replicate in low oxygen environment
• Release toxin which enters bloodstream
• Acts on the nervous system to block
neurotransmitters

20
 Tetanus

• The toxin causes the disease.

• The vaccine consists of toxin which is rendered inactive
(toxoid) but retains immunogenicity.
• The immune response to the vaccine is directed against the
toxin rather than the bacteria.
• Vaccination results in serum antibody production. The
antibody neutralizes toxin.
• Three doses of vaccine recommended to raise antibody levels
to protective levels.
• Immunity lasts about 10 years.
• Vaccination prevents disease but is incapable of disease
eradication.
21
 Neonatal and Maternal Tetanus
• Tetanus in Children has been largely controlled by
vaccination.
• In 1999, WHO estimated 290,000 cases of
neonatal tetanus which resulted in 14% (215,000)
of neonatal deaths. 30,000 (5%) of Maternal
deaths were due to tetanus.
• Target to eliminate neonatal and maternal deaths
due to tetanus by 2005 by vaccination of women
of child bearing age.
22
Diphtheria

23
 Diphtheria
• Corynebacterium diphtheriae.
• Gram positive bacillus.
• Only produces a powerful exotoxin when infected
with a bacteriophage. (the phage carries the toxin
gene which coverts the bacteria from non-toxigenic
to toxigenic)
• Diphtheria is now well controlled by national
immunization programs.
• Only receives attention when routine immunization
programs fall.

24
Diphtheria

25
 Whole Bacteria or Virus
Vaccines

Cholera bacteria
Polio virus
26
 Whole Killed
• Whole pathogen grown and killed
– Heat, chemical modification (formaldehyde,
βPL,..)
– Pertussis, cholera.
– IPV (Inactivated Polio), Influenza, Hepatitis A.
• Advantage
– Relatively easy
– Generally safe to administer - no risk of
reversion, infection.
27
 Whole Killed

• Disadvantages
– Numerous injections normally required
– No or limited cellular immunity
– Immunity often shorter-lasting than live vaccines
– Reactogenicity from LPS, lipids..
– Adjuvant often required (alum, emulsions)
– Risk of growing pathogenic organism (eg. polio)
– Risk of incomplete inactivation
– Inappropriate immune response ? eg RSV ???
– Can not focus immunity on protective antigen.
28
 Inactivated Whole Cell Cholera Vaccine
Manufacturing Process
Fermentatio
n
SBL Vaccine

Cholera toxin
B subunit
Inactivation

Concentratio
n Bulk Formulatio
And Monovalent n
Washing
29
 Subunit
• Purify a protein or proteins from pathogen
– Eliminate reactogenic contaminants (eg LPS)
– Selective presentation of 'protective' antigens
• Pertussis
– Pertussis toxin + filamentous haemagglutinin + pertactin (no
LPS)
• Influenza (subunit)
– Mainly haemaglutinin + neuraminidase
• Disadvantages
– Requires growing the pathogen and purifying
protective (antigens) subunits.
30
Pertussis

31
 Pertussis
• Bordetella pertussis.
• Gram negative coccobacillus.
• Extremely contagious infecting almost all
susceptible close contacts.
• WHO estimated in 1999 that pertussis was still
responsible for about 300,000 deaths mainly
in sub-Saharan Africa.
• 40 million cases annually.

32
 Pertussis Vaccines
• Whole cell vaccine.
– Whole bacterial cells which are rendered non toxic using
formalin.
– Problem with this vaccine was its reactogenicity.

• Acellular pertussis vaccines.

– Contain combinations of purified antigens.
– In the USA, acellular vaccine was introduced for booster
dosing, but in Jan 2000 it was recommended for all doses.

33
 Bordetella pertussis

Pertussis toxin

Pertactin Filamentous haemagglutinin 34

Pertussis components and their role in
protection
• Pertussis toxin: exotoxin secreted during bacterial
growth. Binds to receptors on respiratory tract cells.
• PT is a major virulence factor and is toxic to
mammalian cells.
• Antibody to PT protects mice to lethal challenge.
• Antibodies to PT are elicited after both vaccination
and natural infection.
• PT is a toxin so must be toxoided for use in a vaccine.

35
 Pertussis components
• Filamentous Haemagglutinin: a cell wall component
implicated in attachment to the respiratory
epithelium.
• Antibody to FHA protect mice from lethal respiratory
but not intracerebral challenge.
• Antibody to FHA is produced as a result of infection
or after vaccination.
• Some studies have shown a correlation of antibody
titre to FHA with protection whereas other studies
have not.

36
 Pertussis components
• Pertacin: a 69 kD outer membrane protein (OMP).
• Protects neonatal mice against respiratory
challenge.
• Antibody to Pertactin is produced as a result of
infection or after vaccination with either whole
cell or acellular vaccines.
• Antibodies against Pertactin are implicated in
protection against pertussis.

37
 Pertussis
• Other components being investigated for their
role in toxicity and infection.
– adenylate cyclase
– endotoxin
– tracheal cytotoxin
– heat labile toxin
– agglutinogens

38
 Polysaccharide
• Many bacteria produce a strain-specific capsular
polysaccharide on their surface.
• Antibody to these antigens are protective.
– Streptococcus pneumoniae, Haemophilus Type B,
Meningitis A,C,W,Y (not B!) Typhoid (Vi).
• Can be easily purified.
• Immunogenic in older children / adults.
• But poorly immunogenic in infants
• T-cell independent responses
• Short lived
• Low antibody responses

39
 Different forms of Polysaccharide

LPS Capsular polysaccharide

40
Conjugate
Vaccines

41
Polysaccharide conjugates

42
Haemophilus b

43
Haemophilus

44
 Haemophilus

• Gram negative rods.

• Capsulated or non-encapsulated
• Capsules are polysaccharide.
• Six distinct serological types (a–f) of encapsulated
H. influenzae.
• 95% of all serious infections attributed to type b
(poly ribosyl-ribitol phosphate PRP)
• Most Hib colonization of the upper respiratory
tract are asymptomatic. Disease arises when the
bacteria moves into the blood.

45
 Haemophilus b

• Disease Burden (WHO estimates)

– >500,000 children die from Hib pneumonia per
year.
– >500,000 children die from Hib meningitis per
year.
– 90% of cases are in children under 5 with peak
incidence 6 – 11 months.

46
 Hib Vaccines
• Unconjugated vaccines:
• First Hib vaccines consisted of purified PRP.
• Immunogenic in children >2 years
• Immune response in younger children was poor.
• The response to carbohydrate antigens is T-cell
independent. Young children lack the immune
system maturity required to mount such a response.

47
 Hib Vaccines
• Conjugated vaccines:
• Covalently linking PRP to a protein carrier results in
T-cell recognition of PRP.
• Recruitment of T-helper cells allows production of
antibodies to PRP and importantly induces
immunological memory.
• This type of response is fully developed at a young
age.

48
 Typhoid Vaccine
Vi Capsular Polysaccharide O COOH

O
Fermentatio
n AcO

AcN O

Inactivation

Vi Capsular
Polysaccharid Sterile Formulatio
e Filtration n
Purification
49
Vi Conjugate vaccine
Vaccine against Typhoid Fever

Exoprotein A (rEPA)
C=O

NHNHCO(CH2)4CONHNH
Spacer
C=O

Vi
Capsular Polysaccharide
50
 Live Attenuated

• Most common type of vaccine.

• OPV (oral polio), measles, mumps, rubella, yellow-fever, rotavirus,
influenza, smallpox,
• BCG, typhoid, anthrax,

• Live pathogen selected or genetically modified

causes no disease or only mild disease.
• Derived from non-human source (eg cows, monkeys)
• Selected mild strain from humans
• Passaged (tissue adapted) strain from humans
• Genetically modified (limited ability to replicate)

51
 Live Attenuated
• Advantages:
– Mimics natural infection
– Humoral and cellular response.
– Immunological memory.
– Generally cheap.
• Disadvantages:
– Not suitable for all organisms
– May revert to a virulent form
– Circulating antibody may interfere with response
– Immunity to non-protective antigens
– Safety in immuno-suppressed individuals?
52
Live Attenuated Vaccines

Typhoid bacteria

Measles virus
53
Measles

54
 Burden of Measles

• Measles remains one of the major childhood

killers.
• WHO estimates 30 million cases of measles
per annum.
• 875,000 children die from measles annually.

55
 Measles
• Caused by Rubeola virus
– Genus Morbillivirus
– Family Paramyxoviridae
• Six structural proteins
– 3 complexed to the viral RNA
– Matrix protein associated with inner layer of lipid
membrane
– 2 viral membrane proteins
• Fusion protein – fusion with host cell
• H protein – adhesion of virus to host cell
56
Immune response to Measles

57
 Immune response to Measles
• Antibody response.
– Serum IgM
– Serum IgG
– Serum IgA and secretory IgAs.
• Re-exposure induces a strong secondary IgG
response and prevents clinical disease.

58
 Immune response to Measles
• Cell Mediated Response
– Sensitization of T – lymphocytes.
– Production of cytotoxic T- cells.

• Cellular response is important in recovery

from infection. People deficient in this
response are at increased risk of death.

59
 Measles Vaccine
• Killed Inactivated vaccine was abandoned in
1967 as it did not provide complete
protection.
• An attenuated vaccine was also being
developed and first licensed in 1963. This was
found to be too reactogenic for routine use.
• Further attenuation produced an acceptable
vaccine.

60
 Attenuation of Measles

EDMONSTON B STRAIN
• 24 passages in primary kidney cells
• 28 passages in human amnion cells
• Passages in chick embryo cells
• Vaccine immunogenic but too reactive.
SCHWARZ
• 85 additional passages in chick embryo cells
• Vaccine 90-95% effective, low reactogenicity.
• At least 20 years immunity.
61
Polio Virus

62
 Polio Virus
• An enterovirus belonging to the family
picornavirus.
• Contains RNA surrounded by icosahedral
protein capsid.
• Contains no lipid and is acid stable so can pass
through the stomach.
• Three antigenically distinct types, 1, 2, and 3.
Vaccines contain all three types.

63
Oral Vaccination

64
Response to Polio Vaccination

65
 OPV vs IPV
• Oral Polio Vaccine (OPV)
• Advantages
– Results in production of secretory IgA. Important in
protecting the individual. Reduces the spread of wild polio.
– IgAs has been observed to persist for 5 – 6 years
• Disadvantage
– Genetic instability of Type 3 strain rarely results vaccine
induced poliomyelitis.
– Transmission of vaccine strains to unvaccinated individuals.

66
 OPV vs IPV
• Inactivated Polio Vaccine
– Safe and effective.
– Lack of production of mucosal IgAs results in
failure to eliminate intestinal re-infection and fecal
excretion.
– Recommended for countries where polio is no
longer endemic.

67
 Recombinant
Protein produced by genetic engineering

• Express protective antigen in safe

easy-to-grow organism
– Hepatitis B (HBsAg expressed in yeast)
– HPV (papilloma L1 expressed in yeast)

VIRUS LIKE PARTICLES 68

 Recombinant

• Advantage
– Safe. Growth in non pathogenic yeast cells
– Easier – in case of difficult to grow viruses like
hepB, HPV.
• Disadvantages:
– Need to identify protective antigen/s
– Obtaining antigen in 'correct' conformation
– Usually poorly immunogenic alone
– Poor CMI – requires adjuvant.
69
Hepatitis B

70
Hepatitis B

71
 Hepatitis B
• More than 2 billion people alive have at some time
been infected with Hepatitis B virus.
• 350,000 remain chronically infected carriers.
• Every year there are
– 4 million acute cases
– 1 million deaths
• Carriers
– 25% of children (<7 years) become carriers.
– 10% of older children and adults become carriers.

72
 Aims of Hep B Vaccination
• Vaccination aimed at reduction of:
– Clinical disease.
– HBV transmission.
– Chronic hepatitis and its associated liver damage
and liver cancer.

73
 Hepatitis B
• During infection large amounts of HBsAg are
produced.
• Only small amounts of HBsAg combine with
the cores to form whole virus.
• Remainder is released into the blood stream
as spherical particles or filaments.

74
 Immune Response to Infection
• Antibody to HBsAg indicates clinical recovery
and is associated with immunity to Hepatitis B.
• Antibody to HBcAg is also produced and is first
to appear after infection.
– Present in blood of acute and chronic subjects as
well as those recovered.
– This antibody does not neutralize virus.

75
Plasma derived Vaccines

76
 Plasma derived Vaccines

• Safe and Effective.

• Rely on supplies of infected plasma.
• Long production cycles.
• Fears of contamination with other blood
borne diseases.

77
Recombinant Vaccines

E.coli

S. cerevisiae
78
Recombinant Vaccine

79
 Recombinant Vaccine
• The gene coding for HBsAg was discovered in
1970.
• The gene has been inserted into a yeast cell.
• As the yeast cell grows it produces large
amounts of HBsAg.
• The HBsAg is extracted and purified then
incorporated into the vaccine.

80
 Recombinant Vaccine

• Advantages of the recombinant vaccines.

– Produced more quickly.
– In larger quantities.
– Free from infectious virus particles.

81
 Recombinant DNA modified organisms
Live Vectors

• Cloning of genetic material from one organism

into another.
• The non virulent parent organism expresses
the antigens of the cloned genetic material.
• A vaccine would elicit a response against the
introduced antigen as well as the original
organism.

82
Recombinant DNA modified organisms

Vaccinia virus
expressing papilloma
virus antigens on its
surface.

83
 DNA Vaccines
• Involves the injection of naked DNA coding for
one or more genes.
• The gene is grafted onto another piece of DNA
which acts as a vector.
• Injected into muscle tissue, once in the cell
the gene prompts the cell to produce antigen.
• The immune system then mounts an immune
response.

84
 HISTORY
❑ In 1990, University of Wisconsin, Jon Wolff
found that injection of DNA plasmids produce
a protein response in mice.
❑ In 1993, Merck Research Laboratories, Dr.
Margaret Liu found that intramuscular
injection of DNA from influenzae virus into
mice produced complete immune response
❑ In 1996, trials involving T-cell lymphoma,
influenzae & herpes simplex virus were
started
 DNA vaccines Vs Traditional vaccines

DNA vaccines Traditional vaccines

❑ Uses only the DNA from ❑ Uses weakened or killed
infectious organisms. form of infectious
❑ Avoid the risk of using organism.
actual infectious ❑ Create possible risk of the
organism. vaccine being fatal.
❑ Provide both Humoral &
Cell mediated immunity ❑ Provide primarily
❑ Refrigeration is not Humoral immunity
required ❑ Usually requires
Refrigeration.
 HOW DNA VACCINE IS MADE

Viral gene
Recombinant DNA
Technology Expression
plasmid

Plasmid with foreign gene

 Transform into
bacterial cell

Plasmid
DNA
Bacterial cell
Plasmid DNA get
Amplified
Plasmid DNA
Purified

Ready to use
 METHODS OF DELIVERY
❑ Syringe delivery:-

Either
intramuscularly
or
Intradermally
 Contd..
❑ Gene gun delivery:-

❑Adsorbed plasmid DNA

into gold particles
❑Ballastically accelerated
into body with gene gun.
 HOW DNA VACCINE WORKS

BY TWO PATHWAYS
ENDOGENOUS :- Antigenic Protein is presented by
cell in which it is produced
EXOGENOUS :- Antigenic Protein is formed in
one cell but presented by
different cell
 HOW DNA VACCINES WORK

Muscle Cells
+ Plasmid DNA
 ENDOGENOUS PATHWAY

Nucleus

Plasmid
DNA

mRNA MHC-I

Antigenic
Peptides

Antigenic Protein
 T- Helper Cell

Mu
ltip
ly

Memory T cells
EXOGENOUS PATHWAY

Antigenic Protein come outside

 s e d
o
cyt
ago
Ph

Antigen Presenting Cell

Antigenic Peptides
Memory
T- Helper Cell
Antibodies
Cytokines
Plasma B-Cell

MHC-II
Activated B-Cell Memory B-Cell
WHEN VIRUS ENTER IN THE BODY

Memory T-Cell

Viral Protein

Antibodies
 ADVANTAGES

❑ Elicit both Humoral & cell mediated

immunity
❑ Focused on Antigen of interest
❑ Long term immunity
❑ Refrigeration is not required
❑ Stable for storage
 DISADVANTAGES

❑ Limited to protein immunogen only

❑ Extended immunostimulation leads to
chronic inflammation
❑ Some antigen require processing which
sometime does not occur
 CLINICAL TRIALS
❑ June 2006,DNA vaccine examined on horse
Horse acquired immunity against west
nile viruses
❑ August 2007,DNA vaccination against multiple
Sclerosis was reported as being effective
• Clinical trials to date with naked DNA vaccines
have not proved to be that successful
• DNA vaccines may be useful as a priming dose in
prime-boost regimes due to their ability to induce
cell mediated immune responses.
 Genetic Toxicity

Integration of DNA vaccine into host Genome

Insertional mutagenesis
Chromosome instability
Turn ON Oncogenes
Turn OFF Tumor suppressor genes
 Over Expression of DNA vaccine
Acute or chronic inflammatory responses

Destruction of normal tisues

Generation of Autoimmune diseases

Anti DNA Antibodies

Autoimmune diseases

Autoimmune Myositis
 Antibiotic Resistance

Plasmid used is resistance to

antibiotics for selection

Raise the resistance to same

antibiotic in the host
 FUTURE PROSPECTS

❑ Plasmid with multiple genes provide immunity

against many diseases in one booster

❑ DNA vaccines against infectious diseases such

as AIDS, Rabies, Malaria can be available
 Edible vaccines
• Edible Vaccine involves introduction of selected desired genes into plant
and then inducing these altered plants to manufacture the altered protein.
• Mode of action: Antigen in transgenic plant - Ingestion -Delivered by
bioencapsulation -Taken up by Mcell - Pass on to the Macrophage - IgG,IgE
responses - Local IgA response & Memorycells - Neutralize the attack by
the real infectious agent.
• Potato :
Advantages
Easily transformed.
Easily propagated.
Stored for long periods without refrigeration.
Disadvantage
Need cooking which denature antigen.

107
 • Banana
Advantages
Do not need cooking.
Protein not destroyed even after cooking.
Inexpensive .
Grown widely in developing countries.
Disadvantages
Trees take 2-3 to mature years.
Spoils rapidly after ripening.
• Rice
Advantages
Commonly used in baby food.
High expression of antigen.
Disadvantages
Grows slowly.
Requires glasshouse condition.

108
 Clinical trials
• Norwalk Virus
20 people fed with transgenic potato .
19 (95%)of them expressing Norwalk virus antigen showed seroconversion.
• Hepatitis B
First human trials of potato-based vaccine against Hepatitis B have reported
encouraging results.
The amount of HBsAg needed for one dose could be achieved in a single
potato.
Levels of specific antibodies significantly exceeded the protective level of
10mIU/mL in human.

109
 Pros
• Cost effective.
• Easy to administer.
• Easy to store.
• Acceptable to poor developing country.
• Fail safe
• Activate both mucosal and systemic immunity.
• Heat stable.
• Do not required cold chain maintenance.
• No fear of contamination.
Cons
• Transgenic contamination can occur.
• Antibiotic resistance marker genes can spread from GM food
to pathogenic Bacteria.
• Difficulty in dose maintenance.
110
 Virus-like particle vaccines
• This consist of viral proteins derived from the
structural proteins of a virus.
• These proteins can self-assemble into particles that
resemble the virus from which they were derived but
lack viral nucleic acid, meaning that they are not
infectious.
• Because of their highly repetitive, multivalent
structure, virus-like particles are typically more
immunogenic than subunit vaccines.
• The human papillomavirus (HPV) and Hepatitis C
virus vaccines are two virus-like particle-based
vaccines currently in clinical use. 111
 HPV vaccines are sub-unit vaccines
made of virus like particles: VLPs

Making VLPs: molecular “cut and paste”

HPV 16 L1 VLPs
“cut” the L1 gene from the virus DNA
“paste” into the DNA of another microbe
such as yeast or baculovirus

grow the recombinant microbe in large

amounts
– as it grows it makes the L1 protein

The chemistry of this protein is such that

it self assembles into a virus like particle
- an empty protein shell without DNA

The VLP is morphologically and

immunologically identical to the
HPV virus particle
112
 VLP vaccines: mechanisms

3 key players in the induction

of the antibody response

•Dendritic cells (antigen presenting cells) in the

muscle

•T lymphocytes in the lymph node

•B lymphocytes in the lymph node

 Immune Response to HPV Vaccines:
A Proposed Mechanism1–5

1. Stanley M. Vaccine. 2005 [Epub ahead of print]. 2. Batista FD, Neuberger MS. EMBO Journal. 2000;19:513–520. 3. Tyring SK. Curr Ther
Res. 2000;61:584–596. 4. Roden RB, Hubbert NL, Kirnbauer R, et al. J Virol. 1996;70:3298–3301. 5. Chen XS, Garcea RL, Goldberg I, et al.
MolCell. 2000;5:557–567.
 Th2 cell help for B lymphocytes is crucial
to

•Class switching – determines the type (IgG,etc)

and amount of antibody secreted
dictated by the Th2 cytokines – these in turn
are regulated by
antigen type
antigen dose

•The generation of memory and the efficiency

of the response to recall antigen
under the control of memory Th2 cells
 B cell memory and long term antibody persistence

Amanna et al Imm Revs 2006 211:320

Prophylactic HPV L1 VLP vaccines
Delivered intramuscularly
• rapid access of VLPs to blood vessels
and local lymph nodes
• potent activators of APC
induce good T helper responses for B cells

Efficacy >90% for persistent infection

100% for disease (5 years post
vaccination) in subjects naïve for
vaccine HPV types

Immunogenic high antibody concentrations up to

1000x > than in natural HPV infection

Duration of vaccine induced antibody levels

protection maintained over 5 years

Safe no vaccine related serious adverse

events identified in the trials
to date (70,000 women)
 Nanoparticles in vaccine delivery

•Polymeric nano- and microparticles have recently been shown to possess significant potential
as drug delivery systems.
•Use of biodegradable polymeric nanoparticles with entrapped antigens such as proteins,
peptides, or DNA represents an exciting approach for controlling the release of vaccine
antigens and optimizing the desired immune response via selective targeting of the antigen to
antigen-presenting cells (APCs).
•The efficient delivery of antigens to APCs, especially in dendritic cells (DCs), and the
activation of APCs are some of the most important issues in the development of effective
vaccines.
• Using nanoparticle based vaccine delivery systems, it is possible to target delivery to DCs,
activate these APCs, and control release of the antigen.
•Nanoparticles prepared from biodegradable and biocompatible polymers such as
poly(lactide-co-glycolide) (PLGA), poly(amino acid)s, and polysaccharides have been found to
be effective vaccine carriers for a number of antigens..
118
•Polymeric nanoparticles formulated from biodegradable polymers (PLA, ϓ-PGA) are being
widely explored as carriers for controlled delivery of different agents including proteins,
peptides, plasmid DNA (pDNA) and low molecular weight compounds.
•Controlled delivery systems consisting of nanoparticles can potentially delivery either the
antigens or adjuvants to the desired location at predetermined rates and durations to generate
an optimal immune response.
•The carrier may also protect the vaccine from degradation until it is released.
•Biodegradable polymers provide sustained release of the encapsulated antigen and degrade in
the body to nontoxic, low molecular weight products that are easily eliminated.
•There are three primary mechanisms of adjuvant function: (1) stabilization of antigen, (2)
delivery of antigen, and (3) activation of innate immunity.
•The duration of delivery is likely to affect immunity.
•The biodistribution of nano- and microparticles and the particle-related immune response can
be regulated by controlling the size of the particles.
•Consequently, the size of the particulate delivery system is an important factor for modulating
immune responses via differential interactions with APCs.
•Other potential advantages of the controlled delivery approach include reduced systemic side
effects and the possibility of co-encapsulating multiple antigenic epitopes or both antigen and
adjuvant in a single carrier. 119
Induction of immune responses by nanoparticle-based vaccine

Maturation and activation of DCs by nanoparticles

120
New Technologies and Vaccine
Development
 Issues for Live Attenuated
Virus Vaccines
• Natural infection may not induce
• immunity or optimal immune
responses
• Some viruses cause deleterious
immune responses
• Potential reversion to virulence
– Concern for HIV
• Decreased efficacy due to pre-existing
antibodies
– Influenza
• Decoy antigens on the virus
 Comparison of Vaccine
Technologies
• Live attenuated viruses • DNA vaccines
– Highly effective
– Need for increased potency
– Potential risk
– Designer immune response
– Manufacturing challenge
e.g., Type of TH
• Recombinant proteins
– Specificity: avoid
– Potent antibody response deleterious or diversional
– Non-native forms antigens
– Not induce CTL – Stability
• Viral vectors – Safety
– Risk – Generic manufacturing
– Resistance / pre-existing – Cost
antibody
– Inflammation
 HIV Clade (Strain)
Diversity
H G

J
A

E C

D
B F
HIV and the Pathogenesis of AIDS, ASM Press (1998), J.A.
Heterogeneity of HIV
Strains
 Exogenous Protein Results in
Generation of T Cell Help But
Not CTL
CD4 + Helper
T cell
CD4
Acidified (TH)
Endosome

MHC Class II
Glycoprotein

T cell
Receptor

Exogenous
Endocytosis
Antigen
Golgi
Apparatus
Nucleus
Modified
 DNA
Vaccine
Generation of CTL by DNA
Vaccines
CD8 +
DNA Proteasome
cleaves protein
Cytotox
Vaccine into short peptides ic T
Cytosolic cell
Antigen
(CTL)
CD8

MHC Class 1 Glycoprotein

T cell
Receptor

mRNA
DNA Modified from
Mc Donnell WB and
Golgi Askari FK, NEJM
Nucleus Apparatus 334:42 (1996)
 1918 Flu Pandemic

20 Million Deaths

Courtesy of T Sharrar, Smithsonian Institution

 Initial Demonstration of Efficacy
of DNA Vaccines
• Generation of CTL by DNA vaccine
• Protection by DNA vaccine against
infectious challenge
• Cross-strain protection
H1N1 H3N2
(1934) (1968)

Ulmer JB, Donnelly JJ…Liu MA, Science 259: 1745 (1993)

 DNA Vaccine Protects
Against Cross-Strain
Influenza Challenge
NP DNA Vaccine Control DNA
100

80
Survival

60

40
%

20

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Day After Infection
Fu T-M…Liu MA and Donnelly JJ, J Virol 71:2715 (1997)
 Plasmid Non-Specific
Stimulation
Due to:
•PuPuCGPyPy sequences
– “CpG motifs”
•Potential means to increase / decrease / or
change nature of immunogenicity of
DNA Vaccines

Krieg AM…Klinman DM, Nature 374:546 (1995) Klinman DM…Krieg AM, PNAS 93:2879 (1996)

Sato Y…Carson DA and Raz E, Science 273:352 (1996) Klinman DM…Ishijatsubo Y, JI 158:3635 (1997)
HIV
 HIV
Envelope
gp12
0

gp4
1
Different Forms of HIV Envelope
Used for Immunizations

Monome
r gp120 Soluble Membrane
Oligomer Bound gp160
Recombi gp140
nant DNA vaccine
protein Recombinant
B cell

Antibodies
CD8 +
DNA Proteasome Cytotox
Vaccine cleaves protein
into short peptides ic T
Cytosolic cell
Antigen
(CTL)
CD8

MHC Class 1 Glycoprotein

T cell
Receptor

mRNA
DNA Modified from
Mc Donnell WB and
Golgi Askari FK, NEJM
Nucleus Apparatus 334:42 (1996)
 Clinical Trials of DNA Vaccines

• HIV
– Therapeutic and prophylactic
– Multiple vaccines / multiple trials
• Influenza
• Malaria
– Multiple vaccines / multiple trials
– Antigen + cytokine genes
• Hepatitis B
• Cancer
• (Gene Therapy)
Second Generation DNA Vaccines

• Increased potency

• “Designer” immune response

• Oral delivery
Encapsulated DNA:
Microparticles
DNA Vaccine Replicons Rapidly
Produce More Protein Antigen
Replicon DNA
Nucleus

m
7 3 A
5' Antige (
Replicase nsProteins n
'
G n)

Antigen
mRNA
 “Designer Gene
Vaccines”
Genes Encoding:
Replicon: •Cytokines
•Amplify •Co-stimulatory
molecules
antigen
mRNA •Targeting
molecules

Ç or † CpG Content:
(immunostimulatory
sequences)
Protection of BALB/c mice after immunization with
plasmid DNA and/or recombinant MVA

Immunization 1 Immunization 2 % Protection*

DNA DNA 0

MVA MVA 20

DNA MVA 100

MVA DNA 0

*5 animals/group
Antigens used: PbCSP + PbTRAP
J. Schneider, …, A.V.S. Hill, Nature Medicine 4:397-402
 DNA Vaccines: Tool
for Functional Genomics/Proteomics

Geno
me
In Vitro
Expressi
on
Sele Ligat
ct e
Gene
s

In Vivo
Expression/Func
tion or
Immunogenicity
 Characteristic of DNA
Vaccines
• Able to generate CTL, antibodies, TH
– Cross-strain protective CTL
– Advantages of antigen structure for antibodies
• Transmembrane protein
• Native glycosylation
– TH intrinsically TH 1
• Can co-deliver cytokines to augment or alter T phenotypes
H
– Mechanisms for CTL and TH generation elucidated
– Ability to stimulate desired immune responses not
induced by wild-type disease
– Avoid certain limitations/concerns of viral vectors
Characteristics of DNA Vaccines
• Second generation DNA Vaccines
– Increased potency
– Oral/Mucosal delivery
– Facile manipulation of immune responses
• Potential advantages for clinical usage
– Ability to generate T cell immunity: critical for many
unconquered diseases
– Key characteristics relevant to globally-needed vaccines
• Generic technology
• Stability
• Manufacturing ease
• Cost
• Potential duration of immune response