Vaccine

Vaccine

Definition: Vaccine is a preparation of antigens used for vaccination

Ideal vaccine characteristics:

1) Safe
2) Protective
3) Gives sustained protection
4) Induces neutralization antibody
5) Induces protective T-cells
6) Availability
7) stability
8) Practical consideration

Low cost per-dose

Ease of administration
Few side effects
Vaccination Principle
 Types of Vaccines

1. Live attenuated
2. Inactivated/Killed organism
3. Subunit
4. Peptide
5. Conjugate
6. DNA-based
7. Viral vector
8. Edible
 1. Live Attenuated Vaccine
Definition:
– Live virus,
– antigenically overlap WT
– restricted in pathogenesis
Immunity:
most efficient for both cellular and humoral immunity
Example:
Measles,
Mumps
Rubella
Varicella (chicken pox)
 Live attenuated
• Derived from wild virus
• Attenuated through repeated culturing or recombination
• must replicate
Advantage
• strong cellular immune response similar to natural infection.
Disadvantages:
• severe reactions possible in immunosuppressed vaccinees
• can revert to pathogenic form
• require only one or two doses
• interference from circulating antibody
• require refrigeration or lyophilization (freeze-dry)
• unstable
Mechanism
(b) Recombinant DNA technology
Live Attenuated: Cholera
 2. Inactivated/Killed organism vaccine
• Heat or formaldehyde killed
• Example: Polio, Hepatitis A
• Advantage:
• when no acceptable attenuated microorganism is available
• no reversion to virulence
• enhanced ‘secondary’ response
• Disadvantages
• Immunity is often brief & may require booster dose
• Extreme care is required in their manufacture
• cell mediated response is generally poor
• Some may induce hypersensitivity
 3. Subunit Vaccine

Definition:

A vaccine composed of purified antigenic determinant that is

separated from the virulent/diseases causing organism. Vaccines that
use components of a pathogens rather than the whole organism are
subunit vaccine.

General process of Subunit vaccine production:

1. Traditional method:

2. Synthesis of antigen by gene cloning:

 Subunit Vaccine

Advantage:

1) less contaminant antigen.

2) reduce adverse reactions to the vaccine
3) administrated in more concentrated form
4) Possible synthetic production

Disadvantages:

1) Purification is costly
2) Protein conformation may changed when expressed in prokaryotic
system
Subunit Vaccine: HSV vaccine
 Subunit Vaccine: HSV vaccine

 Killed or attenuated: increase risk of cancer

 HSV-1 gD → cloned → Chinese hamster ovary (CHO) cells -->>

properly glycosylated

 Membrane bound protein (gD) difficult to purify !!

 Remove cTerminal transmembrane binding domain

Subunit Vaccine: HSV vaccine
 Subunit: Foot & Mouth Disease Vaccine (FMDV)

 FMV RNA → cDNA → RE digestion → (VP1 + MS2 replicase) cloned in E.

coli ; 396aa long

 Empty Viral Capsid Lack infectious NA:

 Subunit Vaccine: Cholera
 Vibrio cholerae, secretes hexameric enterotoxin, consists of one subunit,
the A subunit, that has ADP ribosylation activity and stimulates adenylate
cyclase, and five identical B subunits that bind specifically to an intestinal
mucosal cell receptor.
 The A subunit has two functional domains: A1 peptide, which contains the
toxic activity, and the A2 peptide, which joins the A subunit to the B
subunits.
 Traditional cholera vaccine is phenol-killed. Only moderate protection (~3
to 6).
 Recent: heat-inactivated V. cholerae Inaba classic strain, heat-inactivated
Ogawa classic strain, formalin-inactivated Inaba El Tor strain, formalin-
inactivated Ogawa classic strain, and a recombinant cholera toxin B
subunit, has come into use. Taken orally.
 Subunit Vaccine: SARS
 Coronavirus, ssRNA (+), ~30 kb.
 In nature, the viral spike protein, which is inserted into the viral membrane,
binds to a receptor protein on surfaces of mammalian host cells.
 The viral and cell membranes can fuse, and enter into the host cell. The
spike protein (or the external portion of the molecule) is an attractive
candidate for the development of a subunit vaccine.
 External portion of the spike protein (i.e., amino acids 318 to 510) could bind
efficiently to the host cell receptor protein.
 Subunit Vaccine: SARS
 Express a spike 192-amino-acid peptide in CHO cells.
 Additionally, includes a mammalian secretion signal, an N-terminal
(Staphylococcus aureus) protein A purification tag, and a tobacco etch
virus protease cleavage site (Fig).
 The recombinant protein synthesized in CHO cells was secreted into the
growth medium, purified by affinity chromatography on a column containing
immobilized immunoglobulin G, and then digested with tobacco etch virus
protease to remove the protein A purification tag.
 Fully glycosylated form shown to elicit a strong immune response in mice.
Subunit Vaccine: Staphylococcus aureus
 Subunit Vaccine: Staphylococcus aureus
 The gram-positive bacterium cause of hospital-acquired infection.
 Produces a pore-forming toxin.
 Whole-cell attenuated or killed vaccines not been effective.
 Subunit vaccines of individual bacterial surface proteins gave only partial
protection.
 Recent more effective subunit vaccine: by combining several of the
bacterium’s antigens.
 S. aureus, 23 bacterial outer surface proteins were identified from
genomic DNA sequence data. Coding regions & without signal sequences
cloned into plasmid vectors, expressed in E. coli with a poly-His tag at the
N terminus of the protein.
 Among 23, some proteins affording more protection than others.
 A mixture of the four proteins that individually generated the most
effective antibodies
 Subunit Vaccine: Human Papillomavirus

 causative agent of many common STDs.

 Type 16 is associated with approximately 50% of cervical cancers.
 Virus-like particles assembled from the major capsid (L1) proteins of types
6, 11, 16, and 18 was approved used.
 The L1 protein can self-assemble into virus-like particles, immunogenic.
 gene for the L1 protein from each of the four virus types was cloned and
expressed in a recombinant Saccharomyces cerevisiae (yeast) strain.
 4. Peptide Vaccine
• A peptide vaccine is any peptide which serves to immunize an
organism against a pathogen.

• Peptide vaccine are often synthetic and mimic naturally occurring

proteins from pathogens.

• Peptide vaccines incorporate one or more short or long amino acid

sequences as tumor antigens, combined with a vaccine adjuvant.

FMDV VP1:
C-term aa: 141-160, 151-160, 200-213
N-term aa: 9-24, 7-32, 25-41
Carrier: Hemocyanin
 Peptide Vaccine

Advantages
• Readily synthesized and purified at low cost
• Stable in many storage conditions
• Off-the-shelf reagent
• Safe
• Very effective at inducing CD8 or CD4 T cell responses in vivo in
humans
• Enables direct monitoring of T cell responses induced by the
vaccine.
• Safety in many studies
• Using defined epitopes avoids use of uncharacterized antigens
that may have nontherapeutic autoimmune activity
• Repeated booster vaccines feasible
 Peptide Vaccine

Limitations

•Class I MHC restriction limits relevance of individual peptides to

certain HLA types.
•Short peptides may bind directly to MHC on non-professional APC,
which may induce tolerance
•Rapidly degraded by serum/tissue peptidases
•Peptides with low affinity for MHC may be poorly immunogenic
•Patients have variable repertoires for melanoma antigens: a peptide
vaccine may have to include a large number of peptides to be useful
across a wide range of patients.
•Immune responses may be transient and/or of low magnitude
 Peptide Vaccine: Foot-and-Mouth Disease
 VP1
 Carrier: Hemocyanin
 C-term aa: 141-160 (sufficient immunigenic)
151-160
200-213
 N-term aa: (lower levels of antibodies)
9-24
17-32
25-41
 Peptide Vaccine: Malaria
 Plasmodium; cause malaria
 merozoite surface protein 3:
• Highly immunogenic.
• N-terminal (variable strain to strain);
• C-terminal (highly conserved).
 Chemically Synthesized: peptides B, C, and D had a major inhibitory effect
on parasite growth.
 amino acid 181 to 276 of was finally chemically synthesized
 5. Conjugate Vaccine
• Conjugation of the polysaccharide antigen with a protein.

• Necessity: microbial polysaccharides are T‑independent

antigens, which are usually poorly immunogenic
• Advantage:
• offers better and long time protection.
• Disadvantages
• Preparation is more complex and complicated.
 6. DNA-based vaccine

• What it is
• Why or When should go for DNA based vaccine
• How to prepare
DNA Based Vaccine
How it gives protection
- cause the immune system to produce CTL besides Ab
- Functionally, the motifs—CpG oligodeoxynucleotides (ODNs)— can
directly stimulate multiple types of immune cells including
- monocytes
- macrophages,
- dendritic cells (DCs),
- B cells and
- T cells.

What happens in Myocytes:

 DNA-based vaccine

# Advantages
• manufactured far more easily
• DNA is very stable and resists temperature extremes
• Possible to add heterologous epitopes
• cell mediated (Th1 and CTL) and humoral immunity
• conserving the native conformation of epitopes

# Disadvantages/Limitations
• DNA integrates into the host genome
• anti-double stranded DNA antibodies -- autoimmune disease
• Minute amount of DNA may induce tolerance than immunity
 DNA-based vaccine

EXAMPLE: Clinical trial /Ongoing research:

Human:
- influenza,
- herpes,
- hepatitis B,
- malaria,
- several forms of cancer,
- HIV and
- DNA vaccines for some tumors & some allergens
Animal: infectious hematopoietic necrosis virus (IHNV)
 7. Viral Vector Vaccine

Necessity: alternate vaccine strategy

Types of Viral Vector:

1) Adenoviral vector
2) Retroviral vector
3) Adeno-associated viral vector (AVV).
4) Herpesvirus
5) Poxvirus

Disadvantages:
Vector vaccine: Vaccinia virus
 Vector vaccine: Vaccinia virus
 The DNA sequence coding for a specific antigen, such as HBcAg, is
inserted into a plasmid vector immediately downstream of a cloned vaccinia
virus promoter and in the middle of a nonessential vaccinia virus gene,
such as the gene for the enzyme thymidine kinase (Fig. A).
 This plasmid is used to transfect thymidine kinase-negative animal cells
in culture, usually chicken embryo fibroblasts, that have previously been
infected with wild-type vaccinia virus, which produces a functional
thymidine kinase.
 Recombination between DNA sequences that flank the promoter and the
neutralizing antigen gene on the plasmid and the homologous sequences
on the viral genome results in the incorporation of the cloned gene into the
viral DNA (Fig. B). Although the recombination event is rare, the absence of
thymidine kinase activity in the host cells and the disruption of the thymidine
kinase gene in the recombined virus render the host cells resistant to the
otherwise toxic effects of bromodeoxyuridine. This selection scheme
enriches for cell lines that carry a recombinant vaccinia virus.
 The definitive selection of cells with a recombinant vaccinia virus is made
by DNA hybridization with a probe for the antigen gene.
 8. Edible vaccine
• The vaccines are produced in genetically engineered plants and could
provide safe and inexpensive immunization against diseases for which
a protective antigen has been deemed
• generate transgenic potatoes, tomatoes, bananas, or other fruits
and vegetables to protect individuals against diseases like
• tetanus,
• diphtheria,
• hepatitis B, and
• bacterial enteric diarrhea

• Large scale, inexpensive, and safe source of vaccines

•  do not require extensive storage or refrigeration
•  trigger both cell mediated immunity & mucosal immunity.
•  They are nutritious and easy to administer (safe & painless)
•  Multiple vaccinations per plant (in theory)
Edible vaccine
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 mAb Application

Applications:

1.Diagnostic test
2.Therapeutic treatment
3.Autoimmune disease
4.Cancer treatment