2. Viral Immunogens

3. World Health Organization: Eight out of ten deaths are due to infectious agents. Solution: vaccination.

4. Goals of vaccination Control disease: Prevention Reduction of pathogenesis Shorten interval to recovery Reduce transmission/spread Safety, efficacy, economy

5. 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

6. 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

7. 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

8. 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

9. 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)

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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)

18. 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

19. 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

20. 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)

21. 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)

23. 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

24. Duration of the antibody responses of mice to plasmid encoding BHV-1 tgD

25. DNA vaccines: disadvantages Efficacy: humoral immune responses low in target species such as humans, cattle, etc. Safety: no information about long-term effects

26. 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,

27. BHV-1 virion gD gC gB Envelope Tegument DNA Nucleocapsid

28. Effect of immunization with BHV-1 glycoproteins on clinical response and virus shedding in calves challenged with BHV-1/P.haem.

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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.

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

37. Histopathology Results 10 Days after Formulations were Administered in 50 μ l SC

38. 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

39. Methods of delivery Syringe and needle Nasal spray Liquid to drink Needle-free devices (Biojector, Pigjet) Transdermally (patches)

40. Needle-free delivery method: Biojector for all types of vaccines Biojector – Left hip, ID IM SC ID

41. Gene gun immunization for DNA vaccines

42. 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

43. 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

44. 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

45. 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

46. 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