Viral Immunogens

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Viral Immunogens
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World Health Organization

• Eight out of ten deaths are due to infectious
  agents.
• Solution vaccination.

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Goals of vaccination

• Control disease
• Prevention
• Reduction of pathogenesis
• Shorten interval to recovery
• Reduce transmission/spread
• Safety, efficacy, economy

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Vaccination successes

• 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

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Vaccination problems

• 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

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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

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Viral pathogenesis

• 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

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Types of viral vaccines

• Conventional whole virus
• Live attenuated
• Inactivated
• Genetically Engineered whole virus
• Live mutant
• Live replication defective
• Genetically Engineered subunit
• Viral vector (adenovirus, vaccinia virus, herpes
  virus)
• Replicon (Sindbis virus, SFV)
• Plasmid vector (DNA vaccine)
• Subunit (protein, peptide)

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Historical perspectives

• 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

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Live attenuated virus vaccines properties and
advantages

• 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
• Examples
• 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

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Generation of live attenuated virus vaccines
empirical methods

• naturally occurring
• Cowpox, bovine rotavirus for pigs, turkey
  herpesvirus for chickens
• serial passage in tissue culture
• point mutations accumulate
• serial passage in heterologous natural host
• hog cholera in rabbits
• selection of cold-adapted (temperature-sensitive)
  mutants and re-assortants
• unable to replicate well at body temperature, but
  get into nasal cavity at lower temperature

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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

• Virus needs to lose virulence but retain
  immunogenicity
• Inactivating agents
• Formaldehyde
• ß-propiolactone
• Ethyleneimine
• Reliable tests are needed to assure inactivation
• Formulation with adjuvant is needed for efficacy

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Inactivated virus vaccines advantages and
examples

• Advantages
• safety (no spread, revertants or latency)
• relatively easy and inexpensive to produce
• Examples
• 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

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Inactivated virus vaccines disadvantages

• usually only one arm of the immune response is
  stimulated
• (humoral)
• Delay in opnset of immunity and duration of
  immunity short
• antigens may be modified due to the inactivation
  process
• may induce adverse effects, i.e. potentiate
  disease (RSV, FIP)
• strong adjuvants are needed, which may not be
  safe
• 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

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Genetically engineered whole virus vaccines
replication competent

• 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)

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Genetically engineered whole virus vaccines
replication incompetent

• 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

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Genetically engineered vectored vaccines

• 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

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Genetically engineered vectored vaccines
advantages and disadvantages

• Efficacy may be high (antigens made in the host)
• Induction of mucosal immunity possible
• sprays, aerosols, feed, water
• Potential for immunity in ovo
• BUT
• Pre-existing immunity may be a problem
• Safety issues (attenuation of the vector,
  latency, genomic insertion immunosuppressed
  people, stability)

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Plasmid as vector DNA vaccine

• Bacterial plasmid with
• Selectable marker Antibiotic resistance
• Promoter HCMV
• HCMV intron
• BGH poly A
• Vaccine insert
• Built in adjuvant activity (CpG)

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DNA vaccines advantages

• Conceptual Advantages
• 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
• Non-infectious
• Multiple deliveries possible
• Not limited by pre-existing immunity
• Demonstrated potential as vaccine in neonates

• Practical Advantages
• Potential to encode multiple
• antigens
• Stable
• No cold chain needed
• Low development cost
• Low production cost
• No tissue reactions

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Duration of the antibody responses of mice to
plasmid encoding BHV-1 tgD
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DNA vaccines disadvantages

• Efficacy humoral immune responses low in target
  species such as humans, cattle, etc.
• Safety no information about long-term effects

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Genetically engineered subunit vaccines

• Identify protective viral protein(s)
• Identify, sequence and clone gene
• Express gene in prokaryotic (bacteria) or
  eukaryotic (mammalian or insect cells) expression
  system
• Purify protein scale-up
• Formulate protein or peptides in appropriate
  adjuvant or delivery vehicle
• VLPs calicivirus, rotavirus,

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BHV-1 virion
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Effect of immunization with BHV-1 glycoproteins
on clinical response and virus shedding in calves
challenged with BHV-1/P.haem.
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Subunit vaccines advantages and disadvantages

• Advantages
• Safe
• Marker vaccine
• Efficacious
• Examples
• Hepatitis B surface Ag (yeast)
• Herpes simplex gB and gD (CHO cells)
• Fe LV gp70 (E coli)
• BHV-1 gD, gB, gC (MDBK)

• Disadvantages
• Expensive to develop and produce
• Folding and post-translational modifications
  important
• Needs adjuvant which may cause side effects
• Often only humoral immune response is stimulated
• Duration of immunity short

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Synthetic peptides

• Identification of B cell and T cell epitopes
• Peptides synthesized chemically - lt 64 aa
• String of peptides or mixture
• Good adjuvants needed
• Often disappointing results
• Limited epitopes
• Most B cell epitopes are conformational
• Examples FMDV, rabies virus

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Adjuvants

• 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

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Types of Adjuvants

• Freunds 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

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Adjuvants

• 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

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Immune stimulatory molecules

• Cytokines (IL-1,2,4,5,10,12, GM-CSF, IFN-?)
• PAMPS pathogen associated molecular patterns
• ds RNA or poly IC
• unmethylated CpG DNA or CpG oligodeoxynucleotides
  ODNs
• imidazoquinolines

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CpG ODN as adjuvant

• 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-? and
  IgG2a, or a balanced response.

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Formulation of BHV-1 tgD with CpG ODN and
conventional adjuvants in mice cellular immune
responses
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Histopathology Results 10 Days after Formulations
were Administered in 50 µl SC
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Routes of delivery systemic vs. mucosal (many
viruses enter through mucosa)

• Systemic
• Intramuscular
• Intradermal
• Subcutaneously
• Intravenously
• Adjuvants needed
• Alum
• Montanide
• Emulsigen

• Mucosal
• Oral
• Intranasal
• Intravaginal
• Rectal
• Vehicles needed
• Liposomes
• Polylactide-glycolide microparticles
• ISCOMS
• Alginates

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Methods of delivery

• Syringe and needle
• Nasal spray
• Liquid to drink
• Needle-free devices (Biojector, Pigjet)
• Transdermally (patches)

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Needle-free delivery method Biojector for all
types of vaccines
IM SC ID
Biojector Left hip, ID
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Gene gun immunization for DNA vaccines
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Vaccination time and schedule

• Highest risk of viral disease in young animals
  and children
• Most vaccines given in first 6 months of life,
  and repeatedly, but
• Immaturity of neonatal immune system
• Maternal antibodies
• Window of opportunity for infection
• Interval between vaccinations important
• Standard for human vaccines, variable for
  veterinary vaccines

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Long-term immunity

• 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

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Vaccination of mothers

• Advantages
• Safe for newborn
• Increase duration of protection of the neonate by
  maternal antibodies

• Disadvantages
• Live vaccines teratogenic or abortigeneic for the
  fetus, so need to use inactivated vaccines
• Timing difficult

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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 long-lasting protection.
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• 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