Transcript Viral Immunogens

Lecture No. 9. March 2nd, 2004
Viral immunogens
Sylvia van den Hurk
Viral Immunogens
World Health Organization:
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Eight out of ten deaths are due to
infectious agents.
Solution: vaccination.
Goals of vaccination
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Control disease:
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Prevention
Reduction of pathogenesis
Shorten interval to recovery
Reduce transmission/spread
Safety, efficacy, economy
Vaccination: successes
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Vaccination has saved more lives than all other
methods of control of infectious disease combined.
Childhood immunization programs:
 diphtheria, tetanus, pertussus, Haemophilus
influenzae type B,
 polio, measles, rubella, mumps – chicken pox
 Smallpox eradication (1980)
Eradication efforts in progress: BHV-1, PRV, polio,
rabies
Vaccination: problems
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Viruses with large genetic heterogeneity and
quasispecies are difficult targets for vaccination:
 HIV, HCV
Neonatal immunization difficult:
 Bordetella pertussis, RSV, rotavirus
Vaccination in developing countries problematic:
 cost, cold chain, contaminated needles
Cellular immunity and long-term memory often
difficult to achieve
Desired characteristics of a vaccine
Safety and efficacy
 Induction of humoral and
cellular immunity
 Long-term memory
 Mucosal immunity
 Effective in neonates
 Absence of adverse
reactions
 Absence of tissue damage
Practical considerations
 Multivalent, one-shot
 Low development cost
 Low cost of production
 Stable (no cold-chain)
 Needle-free delivery
Viral pathogenesis
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Consider characteristics of the virus for
selection of vaccine type and delivery route:
Cellular vs humoral immunity, or both
Mucosal vs parenteral vaccination
90% of all viruses enter through mucosal
surfaces
IgA – shorter duration of immunity
Types of viral vaccines
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Conventional: whole virus
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Genetically Engineered: whole virus
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Live attenuated
Inactivated
Live mutant
Live replication defective
Genetically Engineered: subunit
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Viral vector (adenovirus, vaccinia virus, herpes virus)
Replicon (Sindbis virus, SFV)
Plasmid vector (DNA vaccine)
Subunit (protein, peptide)
Historical perspectives
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Edward Jenner: smallpox (1798): first use of
naturally occurring live-attenuated smallpox
vaccine - vaccinia
Louis Pasteur: rabies (1885): first use of
inactivated vaccine - dried infected rabbit
spinal cord - 14 daily doses; 9-year old boy
bitten by rabid dog survived
Live attenuated virus vaccines: properties and
advantages
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replicating virus with reduced virulence (balance between
replication to amplify antigen and clinical effects)
induction of both humoral and cellular immunity
long duration of immunity
inexpensive
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Examples:
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Human: polio, mumps, rubella, measles, yellow fever
Bovine: BVDV, BHV-1, BPIV3, BRSV, rotavirus, coronavirus
Porcine: PRRSV, PRV, TGEV, rotavirus
Canine: CPV, CAV, CDV, CPI, rabies
Feline: FHV, FIP, FPV, FCV
Equine: EHV, EIV, EAV
Generation of live attenuated virus vaccines:
empirical methods
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naturally occurring
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serial passage in tissue culture
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point mutations accumulate
serial passage in heterologous natural host
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Cowpox, bovine rotavirus for pigs, turkey herpesvirus
for chickens
hog cholera in rabbits
selection of cold-adapted (temperature-sensitive)
mutants and re-assortants
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unable to replicate well at body temperature, but get
into nasal cavity at lower temperature
Live attenuated virus vaccines: disadvantages
risk of inadvertent infection if insufficiently attenuated (not always
test models available)
 decreased efficacy if over-attenuated
 risk of reversion to virulence
 risk of recombination with wild-type
 heat lability (lifestock production facility)
 contaminating viruses (mycoplasma, BVDV, blue tongue in canine
vaccines)
 adverse effects on fetus in pregnant animals (BVDV, BHV-1)
 latency (herpesviruses)
 unacceptable for viruses such as Ebola, HIV
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Generation of inactivated virus vaccines
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Virus needs to lose virulence but retain immunogenicity
Inactivating agents:
 Formaldehyde
 β-propiolactone
 Ethyleneimine
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Reliable tests are needed to assure inactivation
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Formulation with adjuvant is needed for efficacy
Inactivated virus vaccines: advantages and
examples
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Advantages:
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safety (no spread, revertants or latency)
relatively easy and inexpensive to produce
Examples:
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Human: polio – monkey kidney cells; Rabies – HAV human
diploid fibroblast; Influenza A,B – eggs
Bovine: BVDV, BHV-1, BPIV3, BRSV, rota, corona, , FMDV
Porcine: PRRS, PRV, TGEV, rotavirus
Feline: FHV, FCV, FeLV, FPV
Equine: EHV, EIV, EAV
Inactivated virus vaccines: disadvantages
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usually only one arm of the immune response is stimulated
(humoral)
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Delay in opnset of immunity and duration of immunity short
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antigens may be modified due to the inactivation process
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may induce adverse effects, i.e. potentiate disease (RSV, FIP)
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strong adjuvants are needed, which may not be safe
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cost per dose higher than for MLV; large amount of antigen needed
(1000 – 10000 x)
killed vaccines may be too much or too little inactivated,
which may lead to safety concerns or lack of efficacy
Genetically engineered whole virus vaccines:
replication competent
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Replication competent virus with one or more specific
deletions in non-essential genes: replicates in tissue
culture and has reduced virulence in the host
 TK herpesviruses, gE, gI (PRV), gE (BHV-1)
Same advantages and disadvantages as conventional
attenuated vaccines, but potential for revertants lower
for double mutants
Can be used as marker vaccine, i.e. vaccinated and
infected animals can be differentiated based on
responses to the deleted protein(s)
Genetically engineered whole virus vaccines:
replication incompetent
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Replication incompetent virus with one or more specific deletions in
essential genes:
 only replicates in complementing cells, transformed with the
missing gene(s)
 replicates in the host, but does not enter new cells due to the
absence of a protein essential for entry
 gH herpesviruses (DISC: disabled infectious single cycle)
Advantage:
 Safety
 Presentation to MHC class I and II, so induction of cellular and
humoral responses
 Can be used as marker vaccine
Disadvantage
 Antigen load may not be high enough for efficacy
Genetically engineered vectored vaccines
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DNA viruses: avirulent with gene of interest inserted
 Vaccinia virus (for rabies in wildlife, rinderpest)
 Adenovirus
 Herpesvirus
 Canarypox virus
RNA virus:
 Sindbis virus
 Picornavirus
 Retrovirus
Bacterial vectors
Genetically engineered vectored vaccines:
advantages and disadvantages
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Efficacy may be high (antigens made in the host)
Induction of mucosal immunity possible
 sprays, aerosols, feed, water
Potential for immunity in ovo
BUT:
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Pre-existing immunity may be a problem
Safety issues (attenuation of the vector, latency,
genomic insertion; immunosuppressed people,
stability)
Plasmid as vector: DNA vaccine
HindIII
AvrII
SpeI
PvuII
SnaBI
Bacterial plasmid with:
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Selectable marker :
Antibiotic resistance
Promoter : HCMV
HCMV intron
BGH poly A
Vaccine insert
Built in adjuvant
activity (CpG)
Esp3I
NsiI
Ppu10I
'HCMV IE1
FspI
HCMV intron
Amp
tgD-CD154
gD signal
7104 bps
AflII
PvuII
PstI
PmaCI
BamHI
BsaBI
StuI
SgrAI
BspMI
BHV-1 gD
bCD154
BGH p(A)
PvuII
MluI
BsmI
AsuII
EcoRI
BsgI
SexAI
PvuII
KpnI
NheI
DNA vaccines: advantages
Conceptual Advantages
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Mimic infection by inducing de
novo synthesis of antigens in
target cells
 Antigen presentation by
MHC Class I and II
 Humoral and cellular
responses elicited
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Non-infectious
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Multiple deliveries possible
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Not limited by pre-existing
immunity
Demonstrated potential as
vaccine in neonates
Practical Advantages
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Potential to encode multiple
antigens
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Stable
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No cold chain needed
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Low development cost
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Low production cost
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No tissue reactions
Duration of the antibody responses of mice to
plasmid encoding BHV-1 tgD
Optical density
1.0
ID,1.5 g
IM,1.5 g
0.8
0.6
0.4
0.2
0.0
0
10
20
Weeks after immunization
30
DNA vaccines: disadvantages
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Efficacy: humoral immune responses low in
target species such as humans, cattle, etc.
Safety: no information about long-term
effects
Genetically engineered subunit vaccines
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Identify protective viral protein(s)
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Identify, sequence and clone gene
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Express gene in prokaryotic (bacteria) or eukaryotic (mammalian or
insect cells) expression system
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Purify protein – scale-up
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Formulate protein or peptides in appropriate adjuvant or delivery
vehicle
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VLPs: calicivirus, rotavirus,
BHV-1 virion
gD
Envelope
Tegument
gC
gB
DNA
Nucleocapsid
Effect of immunization with BHV-1 glycoproteins on
clinical response and virus shedding in calves
challenged with BHV-1/P.haem.
40
Total days of virus
shedding
Sick Days
40
30
20
10
30
20
10
0
0
gB
gC
gD
Placebo
Immunogen
KV
gB
gC
gD
Placebo
Immunogen
KV
Subunit vaccines: advantages and
disadvantages
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Advantages:
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Safe
Marker vaccine
Efficacious
Examples:
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Hepatitis B surface Ag
(yeast)
Herpes simplex gB and gD
(CHO cells)
Fe LV gp70 (E coli)
BHV-1 gD, gB, gC (MDBK)
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Disadvantages:
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Expensive to develop and
produce
Folding and posttranslational modifications
important
Needs adjuvant which may
cause side effects
Often only humoral immune
response is stimulated
Duration of immunity short
Synthetic peptides
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Identification of B cell and T cell epitopes
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Peptides synthesized chemically - < 64 aa
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String of peptides or mixture
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Good adjuvants needed
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Often disappointing results:
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Limited epitopes
Most B cell epitopes are conformational
Examples: FMDV, rabies virus
Adjuvants
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Adjuvants, used from the early 1920s to improve
vaccine efficacy
 Prolongation of release of antigen
 Activation of antigen presenting cells
 Attraction of immune cells
Ideal adjuvant
 Induces protective immune responses
 Induces a balanced Th1/Th2 immune response
similar to natural infection
 Minimal side effects
 Easy to use and administer
Types of Adjuvants
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Freund’s adjuvants (complete and incomplete)
 used in early vaccines
 very immunostimulatory
 associated with severe side reactions, can induce sterile
inflammation of joints
Other Mineral oils
 Strong immune response
 adverse side reactions
Metabolizable and non-mineral oils
 safer to use
 low immune responses
Aluminium hydroxide and Aluminium phosphate (alum)
 lisenced for use in humans
 excellent safety records
 low immune response
Adjuvants
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Most conventional adjuvants induce strong Th2-type responses
characterized by a predominance of IL-4 and IgG1
This type of response is associated with certain immunopathological
complications
 Allergy
 asthma
 autoimmune disease
Resistance to certain intracellular infections ie viruses or bacteria such
as Leishmania major is associated with Th1 type immune responses
Induction of strong immune responses is frequently associated with
inflammatory response in the tissue
Aluminum hydroxide: subcutaneous fibrosarcomas in cats
Immune stimulatory molecules
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Cytokines (IL-1,2,4,5,10,12, GM-CSF, IFN-γ)
PAMPS: pathogen associated molecular patterns
 ds RNA or poly I:C
 unmethylated CpG DNA or CpG
oligodeoxynucleotides ODNs
 imidazoquinolines
CpG ODN as adjuvant
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Safe to use
 Well tolerated by humans and other animals,
currently in human clinical trials
Induces a balanced Th1-type immune response,
characterized by a predominance of IFN-γ and IgG2a,
or a balanced response.
Em
Em
Em
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(2
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pG
)/C
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on
-C
pG
(3
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# of cytokine secreting
cells/1,000,000
IFN-
IL-4
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150
Em
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FC
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# of cytokine secreting
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Formulation of BHV-1 tgD with CpG ODN and conventional
adjuvants in mice: cellular immune responses
150
IFN-
IL-4
100
50
0
ad
ju
v
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C
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FC G
A/
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Em
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(3
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(3
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Tissue damage index
Histopathology Results 10 Days after Formulations were
Administered in 50 μl SC
4
3
2
1
0
Routes of delivery: systemic vs. mucosal
(many viruses enter through mucosa)
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Systemic
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Intramuscular
Intradermal
Subcutaneously
Intravenously
Adjuvants needed
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Alum
Montanide
Emulsigen
Mucosal
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Oral
Intranasal
Intravaginal
Rectal
Vehicles needed
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Liposomes
Polylactide-glycolide
microparticles
ISCOMS
Alginates
Methods of delivery
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Syringe and needle
Nasal spray
Liquid to drink
Needle-free devices (Biojector, Pigjet)
Transdermally (patches)
Needle-free delivery method: Biojector for all types
of vaccines
IM
SC
ID
Biojector – Left hip, ID
Gene gun immunization for DNA vaccines
Vaccination time and schedule
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Highest risk of viral disease in young animals
and children
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Most vaccines given in first 6 months of life,
and repeatedly, but:
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Immaturity of neonatal immune system
Maternal antibodies
Window of opportunity for infection
Interval between vaccinations important
Standard for human vaccines, variable for
veterinary vaccines
Long-term immunity
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Infection with wild-type virus when immunity
wanes: subclinical infection and boost
immunity
Re-infection, viremia, target organ infection:
life-long immunity
 IgG neutralizing virus
Vaccination of mothers
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Advantages
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Safe for newborn
Increase duration of
protection of the
neonate by maternal
antibodies
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Disadvantages
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Live vaccines teratogenic
or abortigeneic for the
fetus, so need to use
inactivated vaccines
Timing difficult
WHO goals for vaccine research
New Immunization Approaches:
To define immunization approaches more efficient than
existing ones that are applicable both to existing
vaccines and to diseases for which no suitable vaccine
yet exists.
New Delivery Systems:
To promote the development of vaccines simpler to
deliver than existing ones with particular emphasis on
reducing the number of doses needed to induce longlasting protection.
New Immunization Approaches
•Nucleic acid vaccines
•Mucosal immunization
•Vaccination in the neonatal period
•Combined vaccines
New Delivery Systems
•Controlled-release vaccines
•Improved immunogenicity of subunit vaccines
•Live vectors