Lecture No. 9. March 2nd, 2004

Viral immunogens

Sylvia van den Hurk

Viral Immunogens

World Health Organization:

Eight out of ten deaths are due to infectious agents.

Solution: vaccination.

Goals of vaccination

Control disease: Prevention Reduction of pathogenesis Shorten interval to recovery Reduce transmission/spread

Safety, efficacy, economy

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

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

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

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

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)

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

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

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

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

Generation of inactivated virus vaccines

Virus needs to lose virulence but retain immunogenicity

Inactivating agents: Formaldehyde β-propiolactone Ethyleneimine

Reliable tests are needed to assure inactivation

Formulation with adjuvant is needed for efficacy

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

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

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)

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

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

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)

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)

tgD-CD154

7104 bps

HindIIIAvrII

SpeISnaBI

Esp3INsiIPpu10I

AflIIPvuIIPstIPmaCIBamHIBsaBI

StuISgrAI

BspMI

MluI

SexAIPvuII

NheIKpnI

BsgIEcoRIAsuIIBsmI

PvuII

FspI

PvuII

'HCMV IE1

HCMV intron

gD signal

BHV-1 gD

bCD154BGH p(A)

Amp

Delivery systems viadifferent routes

Nucleases

Plasmid

IM ID IV Mucosal

Endocytosis

Nucleus

mRNAAg

MHC-I

MHC-II

OUTSIDE HOSTINSIDE HOST

Optimizing DNAvaccine features

Overcomingextracellular barriers

Overcomingintracellular barriers

Targeting for effectiveantigen presenataion

Induction of immuneresponses

Optimization fordesired type of immune

response

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

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

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,

BHV-1 virion

gD

gC

gB

Envelope

Tegument

DNA

Nucleocapsid

Effect of immunization with BHV-1 glycoproteins on clinical response and virus shedding in calves

challenged with BHV-1/P.haem.

gB gC gD Placebo KV0

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20

30

40

Immunogen

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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

Synthetic peptides

Identification of B cell and T cell epitopes

Peptides synthesized chemically - < 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

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

Types of Adjuvants

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

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

Adjuvants

Immune stimulatory molecules

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

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.

Formulation of BHV-1 tgD with CpG ODN and conventional adjuvants in mice: cellular immune

responses

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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

Methods of delivery

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

Biojector – Left hip, IDBiojector – Left hip, ID

IM SC ID

Gene gun immunization for DNA vaccines

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

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

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

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.

WHO goals for vaccine research

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