Role of Cytokines in Vaccinology

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Lecture No. 13. March 30, 2004 Role of
Cytokines in Vaccinology Amelia Woolums
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Role of Cytokines in Vaccinology

• Amelia R. Woolums, DVM PhD
• Diplomate, ACVM
• Diplomate, ACVIM
• University of Georgia, Athens GA 30602

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Introduction

• Variety of cytokines impact immune response
• Complex interactions determine outcome of
  infection
• Vaccination can influence cytokine profile and
  outcome of immune response

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Outline

• Brief review cytokines in response to infection
• Influence of vaccination on cytokines
• Adjuvants
• Use of cytokines as adjuvants

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Cytokines and acquired response to infection

• Intracellular pathogens
• Viruses, intracellular bacteria and parasites
• Extracellular pathogens
• Bacteria, parasites, fungi

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Immune response tointracellular pathogens

• Block infection with neutralizing antibody
• IgG, IgA
• Activate macrophages
• Increase killing of phagocytized pathogens
• Increase MHC II expression
• Induce development of TH1 cells

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Immune response to intracellular pathogens

• Induce activation of TH1 cells
• Activate macrophages
• Activate TH1 and CTLs
• Induce activation of CTLs
• Kill infected targets
• Cytokine production

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Immune response to intracellular pathogens

• In summary, want strong TH1 response
• Some TH2 for mucosal antibody

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Response to intracellular pathogens--cytokines

• Cytokines derived primarily from macrophages or
  other APC
• IL-12 NK and T cell activation
• TH1 differentiation
• suppresses IL-4
• IL-1b T and B cells activation
• macrophage, endothelial activation

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Response to intracellular pathogens--cytokines

• Cytokines derived primarily from macrophages
• IL-6 T and B cell expansion
• plasma cell maturation
• IgA synthesis (with IL-5)
• acute phase response
• TNF-a macrophage, endothelial activation

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Response to intracellular pathogens--cytokines

• Cytokines derived primarily from TH1 cells
• IFN-g macrophage activation
• B cell class switching IgG2
• NK, T cell IFN-g production
• T cell IL-2 and IL-2R expression
• decreases IL-4 production

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Response to intracellular pathogens--cytokines

• Cytokines derived primarily from TH1 cells
• IL-2 T, B, and NK cell activation
• TNF-b (LT-a) macrophage activation
• decreased antibody production

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Response to intracellular pathogens--cytokines

• Cytokine derived from other cell types
• IL-15 produced in many tissues
• expansion and activation of
• NK cells
• CD 8 T cells
• gd intraepithelial lymphocytes (IEL)

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• Summary, cytokines specifically important in
  response to intracellular pathogens
• IL-12
• IL-18
• IFN-g
• IL-2
• IL-15

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Immune response to extracellular pathogens

• Opsonizing and complement-fixing antibody
• IgM, IgG
• Neutralizing antibody
• IgG, IgA

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Immune response to extracellular pathogens

• Want appropriate B cell response
• TH2 response to induce class switching, affinity
  maturation

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Response to extracellular pathogens--cytokines

• Cytokines derived primarily from TH2 cells
• IL-4 B cell clonal expansion
• class switching, IgG1 and IgE
• TH2 differentiation
• inhibits TH1 activity
• IL-5 eosinophil expansion
• IgA and IgE production (species vary)

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Extracellular pathogens, cont.

• Cytokines derived primarily from TH2 cells
• IL-10 suppresses macrophage activation
• suppresses NK, TH1 activity
• TGF-b suppresses TH1 activity
• can suppress or activate macrophages

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• Summary, cytokines specifically important in TH2
  responses, antibody production
• IL-4
• IL-5
• IL-6

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Janeway, 1999
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Janeway, 1999
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Other cytokines relevant to vaccinology

• GM-CSF
• produced by TH1, TH2, macrophages
• expansion of bone marrow-derived cells
• granulocytes neutrophils, eosinophils, basophils
• monocytes/macrophages
• granulocyte and macrophage activation

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Other cytokines relevant to vaccinology

• IL-3
• produced by TH1 and TH2 cells
• effects similar to GM-CSF
• acts synergistically with GM-CSF

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Vaccination and cytokines

• Effect of vaccination on immune response is in
  part through cytokines
• MANY studies in mice
• Genetic background may influence results
• BALBc TH2 bias
• C57BL/6 TH1 bias

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Vaccination and cytokines

• Fewer studies in humans, domestic animals
• Results in mice not always duplicated in natural
  hosts
• More research needed in natural hosts

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MLV vs. inactivated vaccines

• Viral pathogens MLV generally induce stronger
  CMI with some antibody
• Killed high levels of antibody, little CMI
• Antigen presentation pathway involved
• Exogenous vs. endogenous
• Cytokines also involved

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MLV viral vaccines

• MLV vaccines
• Infect cells internal processing, MHC I
  presentation
• Activation of CD8 cells
• CD8 cytokines
• IFN-g, IL-2 likely help drive TH1

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MLV viral vaccines

• Role of other cells in response to MLV?
• NK cell activation IFN-g could drive TH1
• Macrophage involvement through ADCC, uptake of
  opsonized virus
• IL-12 and TH1 induction

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NK activation? IFN-g production?
Better CMI (with TH1 bias likely) in response to
MLV viral vaccines
Presentation on MHC I
CD8 activation, IFN-g, IL-2 production
Virus-infected cells
IL-12 production by macrophages?
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Inactivated viral vaccines

• Antibody response with poor CMI typical
• (adjuvants can change this)
• Exogenous uptake by phagocytes
• Presentation primarily via MHC II
• Activation of CD4 with no activation of CD8
• TH2 bias results

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RSV example, MLV vs. inactivated

• Influence of MLV vs. inactivated studied
  thoroughly in context of RSV infection
• Paramyxovirus
• Leading cause of respiratory disease in infants
• Very similar to BRSV in cattle
• Formalin-inactivated vaccine serious
  vaccine-enhanced disease in children

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RSV example, MLV vs. inactivated

• In mouse models, FI-RSV or viral subunit
  vaccination
• Increased IL-4, IL-5
• Decreased IFN-g
• Live virus vaccination
• Increased IFN-g
• Decreased IL-4, IL-5

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RSV example, MLV vs. inactivated

• Calves vaccinated with FI-BRSV produced less
  IFN-g than calves vaccinated with MLV or calves
  not vaccinated

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RSV example, MLV vs. inactivated

• Evidence in several models that inactivated RSV
  or BRSV induces relative TH2 bias
• May be relevant to other viral pathogens
• Relevance to MLV vs. inactivated bacterial
  vaccines unclear

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Adjuvants and cytokines

• Adjuvants can strongly influence response to
  vaccination
• Some adjuvants can induce strong CMI to
  inactivated pathogens
• Nature of adjuvant and impact on cytokine
  responses important

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Alum and cytokines

• Aluminum salts (including alum)
• Used for decades
• Still only adjuvant approved for humans in US
• Induce strong TH2 responses
• Early IL-4 production key
• IL-4 KO mice have TH1 response to alum!
• (Brewer, 1997)

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Alum and cytokines

• Role of inflammation in adjuvant effect of alum?
• Alum OVA, IL-6 KO or TNFR-1 KO mice
• normal to increased TH1 and TH2 cytokines
• IL-6 and TNF-a inhibit response to alum

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Alum and cytokines

• In sheep, efferent lymph contained no IFN-g,
  IL-1b, or TNF-a following alum Taenia ag.
• Confirms work in mice
• TH2 bias
• Proinflammatory cytokines not key to response to
  alum (Brewer, 1998)

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Freunds adjuvant and cytokines

• CFA potent CMI, but severe adverse reactions
• Only used experimentally, and less recently
• IFA (w/o) weaker CMI, but less reactive

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Freunds adjuvant and cytokines

• In mice increased IFN-g, little or no IL-4, IL-5
  and IL-10 to OVA CFA
• Expected response strong TH1 bias
• IL-12 KO mice decreased IFN-g, slightly
  increased IL-4 in response to CFA
• IL-12 induction key to IFN-g response to CFA

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Freunds adjuvant and cytokines

• Role of proinflammatory cytokines
• IL-6 and TNFR-1 KO mice
• GREATER TH1 and IgG2 responses to CFA
• Suggests these cytokines actually inhibit TH1
  response to CFA (Brewer, 1998)

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Freunds adjuvant and cytokines

• Role of proinflammatory cytokines, cont.
• Studies in rabbits suggest IL-1 not required for
  adjuvant effects of CFA

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Freunds adjuvant and cytokines

• In sheep, IFA Taenia ag
• Increased IFN-g in efferent lymph in 1/3
• Weaker TH1 than expected with CFA
• No increase TNF-a, IL-1b
• Increased GM-CSF in 2/3 (Rothel, 1998)
• In humans, IFA melanoma peptide
• Increased IFN-g response to antigen

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Freunds adjuvant and cytokines

• Various models suggest proinflammatory cytokines
  (IL-1, TNF-a, IL-6) NOT critical for effect of
  Freunds adjuvant
• Counter to classical understanding that
  inflammation leads to adjuvant effect
• Induction of IL-12 and IFN-g is important

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Oil/water emulsions and cytokines

• Very common adjuvants in vet med
• Many in human clinical trials
• Little information re cytokines
• MF59 approved in human influenza vaccine in
  Europe
• Induced TH2 responses in BALBc mice

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Saponins and cytokines

• Saponins extracted from bark of tree (Q.
  saponaria)
• Quil A commonly used in vet med
• More purified forms in human clinical trials
• Toxicity can be limiting
• Decrease dose by combining with other adjuvants

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Saponins and cytokines

• In mice, induce TH1,TH2 cytokines, CTL
• Quil A Taenia ag in sheep
• Increased IFN-g in efferent lymph in 3/3
• Both IgG1 and IgG2 increased
• No clear effect due to IFN-g, unlike mice
• Increased IL-8 but not IL-1b or TNF-a
• 
  (Rothel, 1998)

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Saponins and cytokines

• SBAS2 QS 21 MPL in oil/water
• Increased IFN-g (and antibody titers) in human
  malaria trial
• Significant local reaction
• May be limited to therepeutic vaccines
• (Moingeon, 2001)

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ISCOMS and cytokines

• ISCOMS purified saponin phospholipids/cholester
  ol antigen
• Decreases toxicity of saponin
• Maintains or improves immunogenicity
• In mice, induce TH1 and TH2, good CTL
• Can be given by mucosal route

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ISCOMS and cytokines

• ISCOMS induce influx and activation of
  neutrophils, dendritic cells, macrophages, mast
  cells, and lymphocytes
• BUT IL-6 and iNOS KO mice had normal responses
  to ISCOMS
• Proinflammatory activity not necessary

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ISCOMS and cytokines

• In contrast, adjuvant effect significantly
  decreased in IL-12 KO mice
• However, IFN-g KO mice had normal response to
  ISCOMS!
• IL-12 is critical for ISCOM effect, but through
  mechanisms other than IFN-g induction
• (Smith et
  al, 1999)

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ISCOMS and cytokines

• In sheep, matrix without antigen increased IL-6
  and IFN-g in efferent lymph
• Unusual effect of adjuvant without antigen to
  stimulate immunity
• ISCOMs may uniquely induce lymphocyte and APC
  recruitment (Sjolander, 2001)

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ISCOMS and cytokines

• In primate models, ISCOMs induce IL-4, IL-2, and
  IFN-g to co-administered antigen
• In human clinical trials, improved CTL activity
  to influenza subunits
• 
  (Sjolander, 2001)

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ISCOMS and cytokines

• Results from mouse studies comparable to other
  animal, human trials
• Balance of TH1 and TH2 cytokines make saponin and
  ISCOMs attractive
• Induction of IL-12 key to response to ISCOMS

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Other adjuvants

• Monophosphoryl lipid A (MPL)
• Improves TH1 response in adjuvant mixtures
• Nontoxic mutants of cholera toxin (CT), E. coli
  heat labile toxin (LT)
• CT strong TH2 LT more TH1
• Chimeric molecules can have balance of both

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Cytokines as adjuvants

• Cytokines used experimentally as adjuvants since
  1980s
• Much research has focused on immunotherapy or
  therapeutic vaccination for neoplasia
• Results mixed, but many promising results
• Will not discuss today

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Cytokines as adjuvants

• Problems
• Toxicity or adverse reactions
• Local targeting can help
• Short half-life
• Delayed-release carriers or DNA vectors
  expressing cytokine can help
• Often species specific
• Expense may limit veterinary applications

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Cytokines as adjuvants

• MANY mouse studies
• IL-1b, IL-2, IL-6, IFN-g, GM-CSF, and IL-12 most
  often tested
• Usually have expected effects
• TH2 strong antibody, poor CMI
• TH1 strong CMI, with increased antibody over
  antigen alone

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Cytokines as adjuvants

• In calves, IL-1b once or IL-2 five times improved
  in vitro responses to BHV-1/PI3 vaccination
• Some effect on temperature but not clinical
  scores post challenge
• (Reddy et al,
  1993)
• Multiple dose IL-2 requirement not practical
• Lack of clear effect on clinical signs

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Cytokines as adjuvants

• In cattle, BHV-1 subunit IL-1b improved mucosal
  IgA and resistance to challenge over subunit
  alone (Gao, 1995)
• In sheep, IL-1b improved response to some
  parasite vaccines, enhanced disease after others
• (Lofthouse, 1996)

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Cytokines as adjuvants

• Ruminant IL-1b can sometimes enhance response to
  vaccines
• Lack of clear and consistent clinical benefit may
  not justify expense of recombinant cytokines
• Adverse effects and inconsistent results seemed
  to limit human applications

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Cytokines as adjuvants

• BHV-1 expressing bo IFN-g
• Clinical signs similar to wt virus
• No effect on in vitro IFN-g production
• Stable following reactivation
• Value as vaccine not evaluated
• 
  (Raggo, 2000)

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Cytokines as adjuvants

• IL-12
• Focus of recent research
• In human clinical trials of cancer vaccines
• Mixed results, but research ongoing

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Cytokines as adjuvants

• In goats, vaccinia expressing ca IL-12 vaccinia
  expressing CAEV protein
• No effect on IgG isotypes
• Increased IFN-g, IL-4 by lymphocytes in vitro
• No change in IFN-g/IL-4
• 
  (Cheevers, 2000)
• No clear TH1 bias in vitro
• Effect of vaccine on challenge not tested

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Cytokines as adjuvants

• In cats, vaccination with DNA encoding FIV gp140
  fe IL-12
• Complete resistance to challenge in 3/4 cats,
  compared to 0/4 controls
• Both IL-10 and IFN-g expression increased
  relative to controls
• No clear-cut TH1 bias (Leutenegger, 2000)

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Cytokines as adjuvants

• In outbred species, in vitro response to IL-12 as
  adjuvant may not show strong TH1 bias
• In vitro correlate of protection unclear

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Cytokines as adjuvants

• GM-CSF
• Ongoing research in human vaccine trials
• Good results, therapeutic vaccine for Leishmania
  some cancer vaccines

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Cytokines as adjuvants

• In sheep, DNA vaccine expressing GM-CSF
  Echinococcus ag
• Higher antibody titers than vaccine expressing
  IFN-g, IL-4, or IL-15
• Other cytokines no clear effect on antibody or
  lymphocyte proliferation
• No clear TH1 or TH2 bias due to IFN-g, IL-4

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Cytokines as adjuvants

• Compared to protein subunit, GM-CSF increased
  IgG1/IgG2
• IL-4 also gave high IgG1/IgG2
• Similar to mice (TH2 high IgG1)
• IFN-g had no effect on IgG1/IgG2
• Protection against challenge not measured
• (Scheerlinck, 2001)

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Cytokines as adjuvants

• Cytokines in avian vaccines
• Recent work in chickens
• Variety of cytokines show promise
• As adjuvants
• As therapeutics
• (Min et al,2002 Hilton et al, 2002)

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Cytokines as adjuvants, summary

• MUCH research in mice
• Much less in domestic mammals, humans
• In vitro responses in outbred species often do
  not parallel results seen in mice

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Role of cytokines in vaccinology, summary

• In vitro correlates of protection vary for
  different diseases, species
• In vitro correlates do not always predict
  response to challenge

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Role of cytokines in vaccinology, summary

• Optimum interplay of vaccine, adjuvant, and
  cytokine expression should be evaluated
  independently for each host, disease