:

1 18-22 2552

2 :

., PhD. MSc.

ISBN : 000-000-00-0000-0 1 : 500 : 2553 : milletgroup : : 4 2

. . . . 11000 0-2590-3196-9 0-2965-9152http://www.nvco.go.th

MSc. MSc. MSc.

:

: 1 18-22 2552

:

3 :

. , , , , , , , , , ,

2553

4 :

9 2548 1

( )

5 :

9 2548

1 18 - 22 2552 2 course Vaccinology course Advanced Vaccinology course 200 100

1

( )

6 :

. -

.

.

.

.

7 :

. -

-

. ()

. ()

. ()

. ()

Dr. Stephen J. ThomasDepartment of Virology, US Army Medical CorpsCommander-Armed Forces Research Instituteof Medical Sciences (USAMC-AFRIMS)

8 :

3 4 5 61. Vaccinology in national perspective 11

. ()

2. How Vaccine Work2.1 Antigen 19

.. 2.2 Adjuvants 47

.... 2.3 Vaccine delivery system 59

...

3. 3.1 Researcher perspective in vaccine development 73

.. 3.2 Preclinical vaccine development : Dengue vaccine 79

.. Influenza vaccine 87

.. HIV vaccine 97

.. 3.3 Safety and toxicology assessment of vaccine in pre-clinical study 105

.. 3.4 Clinical trial for candidate vaccine Clinical evaluation 113

..

9 :

Endpoints 119

.() . Statistical issues 123

.. 3.5 Candidate vaccines in clinical trials Efficacy and safety of dengue vaccine : Thailand phase 2b 129

.. Tetravalent live attenuated dengue vaccine phase 2 update 133

Dr. Stephen J. Thomas Armed Forces Research Institute of Medical Sciences HIV vaccine 141

. () 3

3.6 Influenza vaccine 149. .

3.7 Allergen vaccine 155. .

3.8 Ethical issues Human ethics 169

. Human ethics 179

.. Professor Emeritus (Epidemiology)

Animal ethics 191..

4.4.4.4.4. 4.1 Industry view on future vaccines for the global community 197

- 4.2 How to scale up the research product to industrial scale? 207

. -

10 :

5.

5.1 Vaccine Regulatory System in Thailand (National Regulatory Authority) 221.. Senior Advisor in Safety, Effectiveness andUse of Health Products

5.2 National Control Laboratory : Role and Responsibility in vaccine 231

6. 6.1 Public perception of vaccination 243

.. 6.2 Existing immunoglobulin in thailand : Rabies immunoglobulin 249

..

6.3 AEFI monitoring and causality assessment 259. () .

6.4 Pharmacovigilance 271. ()

1 282

2 283

() 3 1 285

4 288

11 :

Vaccinology

2381 3 Vaccinology Immunology,Virology, Clinical research, Epidemiology,Microbiology, Lab of Animal science,Pharmacology Engineering

(Public health), (Vaccine delivery) (ExpandedProgram on Immunization - EPI)

. ()

/ EPI Post marketing surveillance Marketing

Vaccinology in national perspective1.

12 :

(Politics) Vaccinology (Program management) Manpower development

2 1798 - 1935 2 Microbiology Immunology (Smallpox, Rabies, Plague, Diphtheria,Pertussis, BCG, Tetanus, Yellow fever) 1955 2 Microbiology Immunology Molecular biology, Nanotechnology 2 (Polio :IPV-OPV, Measles, Mumps, Rubella,Hepatitis B)

Vaccinology

1798 Smallpox 1955 Polio (IPV)1885 Rabies 1962 Polio (OPV)1897 Plague 1964 Measles1923 Diphtheria 1967 Mumps1926 Pertussis 1970 Rubella1927 TB (BCG) 1981 Hepatitis B1927 Tetanus1935 Yellow Fever

Introduction of the firstgeneration of vaccines for humanBefore World War II After World War II

Source : Plotkin SA and Mortimer EA, 1994.This list is not exhaustive

30

(EPI) 95

13 :

.. 2520 - 2524 : 5 (BCG, DTP, OPV, T, Ty) 5 DTP OPV 2 3, 4 5 .. 2525 - 2529 : (M) 9 - 12 (R) 6 DTP, OPV 3 .. 2530 - 2534 : DTP OPV 4

.. 2535 - 2539 : (HB) (JE) DTP, OPV 5 .. 2540 - 2544 : (M) 1 -- (MMR) JE vaccine 3 .. 2545 - 2549 : (T) dT .. 2550 - 2554 : DTP -HB combined vaccine Influenzavaccine National program Dengue vaccine

vaccines provided in public sector EPI in Thailand 1977-2011

2007-2011projected*BCG x 2

DTP-HB x 3OPV x 5

M or MMRMMRHB x 1JE x 3dT x 3

Influenza?Dengue?

800

2520-24 2525-29 2530-34 2535-39 2540-44 2545-49 2550-542002-2006

.........................BCG x 2DTP x 5OPV x 5

M or MMRMMRHB x 3JE x 3dT x 3

-

1997-2001.........................

BCG x 2DTP x 5OPV x 5

MMMRHB x 3JE x 3TT x 3

-

1992-1996.........................

BCG x 2DTP x 5OPV x 5

MR x 2HB x 3JE x 2TT x 3

-

1987-1991.........................

BCG x 2DTP x 4OPV x 4

MR--

TT x 2Typhoid

1982-1986.........................

BCGDTP x 3OPV x 3

MR--

TT x 2Typhoid

1977-1981.........................

BCGDTP x 2OPV x 2

----T

Typhoid

Source : EPI/GCD/DDC/MOPH,April 2005*Subject to ACIP & DDC considerations, and resource availability

14 :

Dengue, Malaria, HIV, Pneumococcal,

Hib, Rotavirus, Shigella, Leptospirosis : (Dengue Malaria)

Emerging infectious disease (EID)vaccine : Nipah 2542 SARS

Ebola : Chikungunya :

Dengue vaccine Dengue vaccine Chikungunya

Adolescents and adults :

Elderly and the ill :

Influenza vaccine

Occupation : Rabies vaccine, Tetanus vaccine

Tourism : Yellow fever vaccine, Meningococcalvaccine

Post-exposure and treatment : Rabies vaccine, HIV vaccine

Anti-bioterrorism :

Anti-cancer : HPV vaccine

Pandemic preparedness : Influenza vaccine

JE vaccine HepatitisB vaccine

JE vaccine 3 2544 JE vaccine EPI JE vaccine 2513-2523

15 :

Three pandemic waves : weekly combined influenza and pneumonia mortality,United Kingdom, 1918-1919

(AFRIMS) JE vaccine Nakayama clinical trial immunogenicity safety 2523-2533 JE vaccine Nakayama Beijing JE vaccine Nakayama Mouse brain inactivated vaccine JE vaccine 8 2543 clinical trial

Vaccinology

HB vaccine 2535 HB vaccine Plasmaderived HB vaccine Combined vaccine

Vaccinology Influenza A H1N1

Influenza A H1N1 ( 18 .. 2552) pandemic 1918

Death

s per

1,000

pers

ons

6/29 7/27 8/24 9/21 10/19 11/16 12/14 1/11 2/8 3/8 4/5

51015

20

2530

01918 1919

1

2

3

16 :

3 3 2 3 2-3 H1N1 2 1918 host

2 Vaccinology Vaccinology in Public HealthEmergency

Vaccinology Pandemic

Public health , Seasonal influenza vaccine

17 :

Influenza vaccine

2550 Seasonalinfluenza vaccine Pandemic Influenza vaccine Avian flu 2546 2551

2552 (COPD, Asthma, Heartdisease, Cerebro-vascular disease, Renal failure,DM) Influenzavaccine Pandemicvaccine cell culture, adjuvant (Live attenuatedvaccine)

Pandemic vaccine

18 :

6 6

20 ASEAN Forum Vaccine development and production ( DTP, JE, HB ) Vaccinology

19 :

Dr. Edward

Jenner 19 Dr. Jenner (smallpox vaccine) (cowpox) vacca

immunology T cells B cells

2

1) Innate immune system macrophage, NK cell

2) Adaptive immune system Tcel ls (cel l mediated immuneresponse) B cells (humoralimmune response)

adaptiveimmune system memory cells (secondary stimulation) 2 innateimmune system adaptive immune system ()

2.1 Antigen..

How Vaccine Work2.

20 :

adaptive immune system B cells T cells receptor Bcells immunoglobulin receptors Tcells T cell receptors (TcR) B cells T cells T cells MHC(Major Histocompatibility Molecule) HLA (Human LeucocyteAntigen) HLA genetic variation HLA common HLA HLA

immunogenicity immunogenicity immunogen()

1.

(foreigness) (foreign substance)

2. phagocyte hapten immune response allergy penicillin hapten conjugate

3. (chemicalcomposition and complexity) protein, carbohydrate,nucleic acid lipid

4.

21 :

phagocytosis MHC MHC T cells

5. protein, polysaccharide,nucleic acid lipid

Protein : immunogen glycoprotein lipoprotein genome reversevaccinology reverse vaccinology carbohydrate fatty acid lipid conjugation glycoprotein lipoprotein Hib conjugated vaccine polysaccharide conjugate

Polysaccharide : immunogen repeatedepitope epitope ( T cells,B cells)

epitope memorycells (conjugation) memory cells

Nucleic acid : immunogen lipid DNA DNA (single strand DNA) single strand DNA folding double strand DNA double helix combind

Lipid : non-immunogenic hapten lipid adjuvant adjuvant

4 1 1. Primary structure () amino acid polypeptide chain2. Secondary structure () polypeptide ( helixform) ( pleated sheet form)

22 :

3. Tertiary structure () helix pleated sheet 4. Quaternary structure () 2 polypeptide 2 polypeptide

dimeric proteinmolecule

T cells B cells

immunogenicity

1. (Genotype ofrecipient) HLA

2. pool B cells

(infant) (adult) (aging) pool B cells modification

1 :

23 :

3. (dose) (route of administration) tolerance needle free () intranasal,orally transcutaneous mucosal polio vaccine OPV

4. adjuvants immunogenicity

2

1. T-independent Antigen

B cell T cell epitope (repeated epitope)

polysaccharide 2(A) B cell polyclonal activation memory B cell IgM IgG IgE isotype switching epitope phagocyte immunogenicity adjuvant modification adjuvant

2. T-dependent Antigen T cell epitope (antigenic determinant) 2(B) memory B cell isotype switching IgG IgE

2 : epitope (A) T-independent antigen (B) T-dependent antigen

(A) T-independent antigen (B) T-dependent antigen

24 :

T-independent antigen T-dependent antigen B cell 3 naive B cell (B cell resting B cell) naive B cell variable portion constantportion

VDJ VDJjoining recombination DNA variable portion constant portion heavy chain signal heavy chain IgM, heavychain IgG heavy chain IgE T-independentantigen T cell naive B cell transcription VDJ constant S heavychain IgM translation protein IgM IgG IgE constant portion IgG IgE constant portion IgM transcription IgM IgM

primary stimulation plasma cell IgM

naive B cell T-dependentantigen helper T cell (CD40 ligand cytokine) switching looping constant portion recombination transcription transcription VDJ S S IgG looping IgM IgG IgE looping IgM IgG IgE IgG memory cell T-independent antigen T-dependentantigen

Superantigens T cell 1 10,000 1 100,000 (1:104-1:105) monoclonal oligoclonalresponse immune cell superantigen T cell 1 4 1 10(1:4-1:10) polyclonal response superantigen MHC binding ligand

25 :

Epitope (Antigenic determinants)epitope

immune cell B cell T cell

B cell - epitope epitope B cell conformational form (secondary, tertiary quaternary structure) denatured form epitope

protection epitope immune response neutralization ( protection) epitope protection

T cell - B cell T cell conformational form HLA (MHC) (antigenpresenting cell) phagocytosis epitope peptide chain

3 : naive B cell T-independent antigen T-dependent antigen

26 :

(denatured form) binding HLA binding groove T cell

binding epitope HLA 4 binding binding groove

4 : binding groove epitope bind HLA Class I Class II

5 : T cell receptor (TcR)

ReceptorsReceptors T cell T cell

receptor (TcR) , , chain chain chain T cell chain mucosal immunity regulation immune response 5 chain variable (V) V

V

T cell receptor MHC T cell 8-15 amino acid MHC T cell TcR chain CD3

binding groove HLAClass I HLA Class II bindinggroove Class I Class II Class I 8 amino acids Class II 15 amino acids

turn on CD28 co-stimulatory molecule

27 :

6 helper T cell (CD4+) tertiary structure(conformational form) phagocytose antigen presenting cell(APC) dendritic cell, macrophage Bcell present epitope APC epitope present TcR immune response epitope present CD4+ APC HLA class II

T cell MHC binding peptide epitope Tcell excess of different MHC-binding peptide immune response APC

T cell T cell

B cell receptors (BcR) B cell ( surface immunoglobulin membrane immunoglobulin) IgM IgD immunoglobulin immunoglobulin CD3 T cell response 7B cell epitope 4-8residues amino acid epitopes conformational form denatured form epitope conformational form B cell denatured form B cell conformational form denatured form epitope

6 : T cell immune response

28 :

cysteine HEL epitope (syntheticpeptide) 2 aminoacid epitope HEL open loop amino acid 64 80 closed loop cysteine 64 80 disulfide epitope HEL (block) closed loop (% inhibition) HEL open loop (% inhibition) B cell conformational epitope

7 : B cell receptor (BcR)

binding 8 Hen egg-white lysosome (HEL) epitope amino acid 64-80 folding loop loop disulfide amino acid 64 80

8 : conformational epitope block

29 :

B cell epitope linear form conformationalform 9(A) conformationalepitope form complex denature epitope antigenicdeterminant epitope B cell epitope immunogenicity conformational 9(B) epitope 2 B cell epitope folding denaturation epitope

linear epitope B cell linearepitope 9(C) nativeform epitope proteolysis () antigenic determinant (epitope) conformational form in vitro() in vivo () in vivo (expression)

9 : epitope (A) conformational determinant (B) linear determinant (C)neoantigenic determinant binding

30 :

epitope B cell epitope epitope density

B cell epitope denatured form conformationalform

proliferation B cell cytokine T cell B cell migrate lymphoid follicle collaboration T cell proliferation shift isotype IgG IgE T cell B cell 1

1 secondary immune response memory cell (primary immunization) memory cell boost (repeatimmunization) shift IgG memory B cell memory cell 20

Hapten-carriers hapten

hapten

hapten conjugate carrier hapten-carriers hapten-carriers immune response hapten, carrier hapten-carriers 10 B cell hapten conjugate carriers process epitope carrier process carrier carrier peptide MHC molecule T cell carrierpeptide hapten T cell carrier peptide

1 T cell B cell

Native protein Native protein + +Native protein Denatured protein - +

Primary immunization Secondary immunizationSecondary immune response

Antibody production Cell-mediated response

31 :

hapten conjugate carrier Penicillin hapten bind Penicillin allergic Penicillin bind

T cell ( cell-mediatedimmune response)

endocytosis (antigen uptake) T cell process antigen presentation 2

1 HLA Class II CD4+(helper T cell) 11 ()

10 : B cell T cell hapten-carrier

2 HLA Class I CD8+(cytotoxic T cell) 11 ()

soluble antigen cytotoxic Tlymphocytes (CD8+) cross priming cross priming HIV HIV HIV (protection) cross priming 2 TAP protein TAP protein TAP protein HLA I TAP protein

32 :

process HLA I TAP I TAP II TAP-dependent endoplasmic recticulum TAP TAP-independent TAP protein

mucosal dendritic cells process innate immunity adaptive immunity lymphoid organ lymph node dendritic

11 : T cell Class I MHC pathway Class II MHC pathway

cells lymphoid organ process cytotoxic T cell helper Tcell CD8+ T cells CD4+ T cells T cell lymph node (lymphoid tissue) 12

CD1 A, B, C, D E HLA Class I glycolipid cytotoxic Tcells HLA

33 :

CD1

T cells antigen presentation, epitope co-stimulatory molecule CD28 B7 13 co-stimulatory molecule signal T cell receptor

12 :

T cell 1) B7 2) CTLA-4

inhibitory receptor CD28 CTLA-4 B7 suppression immune response

34 :

B cell ( humoralimmune response)

14 B cell () protection

- (neutralization)

- (opsonization) (phagocytosis)

- antibody-dependent cellularcytotoxicity

-

13 : / T cell

complement , phagocytosis complementfragments (inflammation)

epitope

35 :

- T cell Class II linear epitope 12-15 amino acids hydrophobic groove MHC Class II MHC Class I 8-10 amino acids

- B cell() 4-8residues linear form conformational form

14 : B cell ()

- Conventional vaccinology

immunogenicity identifiedgene clone (animal model) immunogenicity 15 ( 5-15 )

36 :

- Genome-based approach (sequence) 16 genome sequence

* DNA microarrays

* in vivo expression technology (IVET) animal model potentially genome

* signature tagged mutagenesis (STM) mutation label mutation

15 : Conventional Vaccinology

mutant mutant antigen pool whole genome

* in silico analysis

* proteomics technology two-dimensional gelelectrophoresis massspec-trometry proteinexpression

high throughput expression in vitro in vivo 15

37 :

- Reverse vaccinology openreading frame (ORFs) 17 2,158ORFs in silico outer membrane protein (surfacemembrane protein)

16 : Genome-based approach

17 : Reverse vaccinology

600 ORFs 600 ORFs clone library 350ORFs (express) clone immunogenicity 15 ORFs 1-2 immunogenicity

38 :

animal model

7 1. Killed whole organisms2. Attenuated organisms

reverse live attenuated measles,rubella polio (OPV)

3. Toxoids toxoids diptheria tetanus

4. Conjugate vaccines polysaccharide B cell memory B cell polysaccharide epitope ( T-independent antigen) conjugation carrier T-dependent antigen carrier T cell plasma cell

polysaccharide(polysaccharide-specific plasma cell) memory B cell conjugate vaccine H.influenzae type b, Pneumococcus Meningococcus

5. Subunit vaccines6. Reverse vaccinology (genomics-

based vaccines)7. DNA vaccines and replicons

DNA intramuscular adjuvant cytosine-phosphorothiolated guanine (CpG) sequence carrier

humoral immuneresponse (B cell) B cell (infant) (elderly) B cell (mature)

- B cell CD21 marginal zone B cell

39 :

(differentiation) B cell B cell naive B cell differentiation memory B cell memory B cell naiveB cell memory B cell plasma cell B cell 18 () naive B cell pool memory Bcell memory Bcell

- naive B cell naive B cell

memoryB cell pool naive B cell memoryB cell 18 () naive B cell (specificity) naive B cell (antigen retention) adjuvant (route ofadministration) plasma cell

18 : naive B cell memory B cell

40 :

1. immunogenicity 2. T cell B cell T cell CD4

CD8 HLA class II HLA class I

3. native of infection (intracellular) (extracellular) conformational form linear form denatured form

4. B cell naive B cell memory B cell

41 :

adjuvant

,adjuvant , side effect adjuvant,adjuvant , adjuvant adjuvant

2 1. (innate immunity)

phagocyte, macro-phage, neutrophil

2. (adaptiveimmunity) 50 specific immune response innateimmunity adjuvant

sentinel cells

mast cells, dendritic cells macrophages innate cells cytokine

2.2 Adjuvants....

How Vaccine Work2.

42 :

(receptor) DNA RNA PatternRecognition Receptors; PRRs lipopolysaccharides; LPS ,tissue trauma stress necrosis

vasoactive molecules, cytokines, chemokines acute inflammation innate cells

cell-mediated immunity (CMI),interferon macrophages neutrophil innate cells sentinel cells pattern

Type I IFNs

Mac.activation

Mac. activation

Neu.& Mac. activation

1) Phagocytosis2) Opsonization,complement activation

Opsonization,complement activation

Molecular patterns of microbesMolecular pattern

of microbesSource

Pattern recognitionreceptors

Principle innateresponse

Adapted from Abbas et al., 2000

dsRNA-activated kinase

LBP/CD14/TLR

TLR/unknown receptor

N-formylmethionylpeptide receptors

1) Macrophage mannosereceptor2) Plasma mannosebinding lectin

Plasma C-reactive protein

dsRNA

LPS

Unmethylated CpGnucleotides

N-formylmethionylpeptides

Mannose-rich glycans

Phosphorylcholineand related molecules

Replicating virus

Gram vebacterial cell wall

Bacterial DNA

Bacterial proteins

Microbial glycoproteinsor glycolipids

Microbial membranes

43 :

Pattern B cells Tcells host cytokine systemic sickness behavior acute phaseprotein response

innate cells specific cells secondary lymphoidtissue B cells

co-stimulatory signal APC draining lymphnode specific cells bone marrow Tcells thymus secondary lymphoid tissue

T cells peptide peptide Antigen Presenting Cells; APC

T cells APC draining lymph node APC antigen signal 1,

44 :

specific cells ,signal , present antigen clonal selection clonal expansion (clone) clone helper T cells cytotoxic T cells effector cells

memory cells clonalexpansion memory cells bone marrow secondary lymphoid tissue

2 stranger model danger model APC draining lymph node

Stranger model : pattern pathogen pattern recognition

45 :

receptor pathogen PAMPs; Pathogen Associated MolecularPatterns signature tag recognition receptor Toll-like receptor (TLR)

Danger model : Professor Matzinger DAMPs;Damage Associated Molecular Patterns

DAMPs innate cells APC maturationprocess draining lymph node T cells

2 draining lymph node

Signal 1 : T cell receptors helper Tcell, CD4 positive cell peptide present MHC class II (MajorHistocompatibility Complex) HLAII (Human leukocyte antigen)

46 :

antigen specific cell lymphocyte B T cells

Signal 2 : T cells phenotype helper T cell type I CTL (CytotoxicT lymphocytes) CMI T cells T cells CMI surface molecule signaling molecule cytokine pattern recognition receptor (PRR) pattern innate cells

screen specific cells

adjuvant proteinantigen signal 1 enhancesignal 2 signal 0 pattern APC draininglymph node present antigen signal0

B cells conformationalepitope T cells APC 2 memory effector cells memory cells

47 :

effector phase effector cells memory cells

protective mechanism presentantigen T cells pathway exogenous pathway MHC class II helper T cells

CMI protective cytotoxic T cells pathway present MHC class I pathway cytoplasm

CMI CTL CTL infect pathway II pathway CMI

48 :

CTL cross presentation exogenous antigen MHCclass I pathway adjuvantphagosome cytoplasm cytoplasm class I subunitvaccine ( ) CMI CTL adjuvant pathway presentation class I CTL

Helper T cells (Th) CD4+ macrophage, CTL Th1 phenotype Th1 cytokine CMI mast cell eosinophil activity CTL macrophage mast cell toxicsubstance Th2 Th17 neutrophil Th9 review mast

49 :

cell modurator subset Th2 natural regulatory

T cells, adaptive regulatory T cells T cells regulatory cells - - - - effector cells, regulatorycells

AdjuvantAdjuvant

adjuvare , adjuvant 2463 titer

2473 adjuvant Freund Freunds completeadjuvant (mineral oil + water + mycobacteria) mycobacterium

adjuvant Professor Janeway adjuvant the immunologists

dirty little secret contaminate LPS adjuvant heterogenouscompounds

Adjuvant 1) Depot effect : adjuvant

particle soluble antigen phagocytic cell localization APC

2) enhance magnitude Th1, Th2 (HMI) (CMI) CMI HMI adjuvant definesignal signal 2

3)

50 :

pattern APC

Adjuvant Type A : Act on signal 0and indirectly on signal 1& 2APC T cells signal 0, 1, 2adjuvant signal 0 pattern patternrecognition receptors (TLR) enhance signal 1 2

Type B: Targeting APCs or favouring Agcapture (enhance Ag presentation) APC helper T cell B cells

Type C : Enhance signal 2 adjuvant

adjuvant

Group A : facilitate Ag uptake, transport enhancepresentation

Group B : Depot effect oil prolong Agpresentation

Group C : signal 0 (PAMPs) LPS, mycobacteria, yeastextracts, ISCOMs enhancesignal 1 2

Group D : danger signal (DAMPs) oil emulsion, alum

51 :

Group E : recombinant Ag cytokine, costimulatory molecules adjuvant

Classification of adjuvants according to immunological events

Gr. Concept of action Examples Key eventsA Facilitate Ag uptake, ISCOMs, Quil A, Alum, Liposomes, Ag localization in the

transport and presentation Polyphosphazine lymph node by APCs

B Depot effect Oil emulsion, Alum (?), gels, Prolonged Agmicrospheres, non-ionic presentation

block copolymersC Signal 0 (PAMPs) Complement, CpG, LPS, mycobacteria, Signaling of PRRs on

yeast extracts, ISCOMs? innate immune cellsD Danger signal (DAMPs) Oil-emulsion surface active Tissue destruction/stress

agents, Alum, IFNs, hspsE Recombinant signal 2 Cytokines, costimulatory molecules APC polarlization, T & B

cell help

Adapted from Schijins, V. 2000. Curr. Opin. Immunol. 12: 456-463

52 :

1 : Particulate adjuvants aluminium salt, oilemulsion, l iposomes, nanoparticles,microparticles ISCOMs

Aluminium salt : particulate adjuvant alum adjuvant aluminiumhydroxide aluminium phosphate alum aluminium salt alum aluminium potassium sulphate alum 2469 2551 Mode of action : aluminum and calciumsalt1. Antigen depot (prolong antigen release) alum prolong Ag release alum 2551-2552

particulate 2. Enhanced Ag uptake (particulate nature)3. Immunostimulant (Alum but not Calciumsalt) alum innate immunity APC Ag processing presentation co-stimulatory molecules signal 1 2 celldamage APC chemokine complement 4. Destabilization of protein antigens present T cells Explaining alum : immunologists dirtylittle secret

2551 alum uric acid damage associated molecular pattern(DAMPs) alum signal 1 2 aluminium salt :1. Th2 Th1 IgG isotype IgE aluminium salt monosodium urate

53 :

aluminium salt cytokine interleukin 4 (IL4) master regulator Th2 Ag, APC, cytokine (IL4) Th2 response Th1 Th1 Th2 2. 4oC -20oC freeze dry antigen adjuvant 3. 4. CMI, CTL5. Th2 IgE CTL humoral immunity

Oil emulsion : oil aqueous surfactant emulsion oilemulsion adjuvant potency alum oil MF59 (Montanide)

oil emulsion adjuvants - oil-in-water (o/w)

draining lymph node MF59

- water-in-oil (w/o) (depot effect)

- water-in-oil-in-water stability

Liposomes : lipid bilayers cell membrane (unilamella) multilamella liposomes liposomes lipid bilayers cytoplasm CTL class Ipathway pathway II liposomes CTL hepatitis A vaccine

54 :

therapeutic cancer vaccine archaeosomes liposomes bacterialike organism

Nanoparticles Microparticles : 10-100 nm nanoparticles 1-100 m microparticles biocompatible, biodegradablepolymer microparticles 10 m formulation long term release release immunomodulator 2

ISCOMs (Immunostimulating complex) : (a cage like structure) saponin, cholesterol, phospholipids strong Th1, Th2 CTL saponin membrane cytosolic pathway adjuvant commercial vaccine icosahedral innate cell icosahedral CMI

Alum StrongTh2, IgE + - - +STw/o emulsion Weak Th1, Th2 - - - / + (peptide) +++STo/w emulsion Weak Th1, Th2 + +++ - -ISCOMs StrongTh1, Th2 +++ ++++ ++++ -Liposomes - ++ +++ ++ -Microparticles< 10 um - ++++ - - -> 10 um - - - - +++LT

Characteristics of particulate adjuvantsAdapted from Cox and Coulter. 1997. Vaccine. 15: 248-256.

Adjuvant Immunomodulation Targeting Presentation CTL induction Depot

Note : ST : short term, LT : long termImmunomodulation : the ability to modify the cytokine networkTargeting : delivering of antigen to the APCPresentation : preserve the conformational integrity and to present to an appropriate effectorsCTL induction : delivering of antigen to the endogenous pathway

55 :

2 : Non-particulate adjuvants mediator PAMPs; pathogen associated molecularpattern particulateadjuvant particulate adjuvants signal 2 polyclonalactivation antigenspecific non-particulateadjuvants

Bacterial products and derivatives : mycobacterium species Freund LPS TLR4 TLR4 agonist TLR4 LPS lipid A LPS toxic innate cells MPL (Monophosphoryl lipid A) Salmonella minisota strong Th1response

Corynbacterium spp, Bacterial toxin

adjuvant cholera toxin,E.coli heat labile exotoxin mucosal tissue bacterial DNA immunostimulatory DNA sequence DNA pattern DNA unmethylated CpG dinucleotides adjuvant DNA Th1 antibody response

Muramyl dipeptide (MDP) and derivatives: mycobacterium subunit muramyl dipeptide Wax Dunit mycobacterium hydrophilic derivative MDP Th2 lipophilic Th1 adjuvant

adjuvant particulate non-particulate

56 :

Saponin : adjuvant water soluble toxic adjuvant hemolysis saponin adjuvant

cytokines, complements carbohydrate polymer

Cytokines : IL-12 commercial form

adjuvant carrier, depot immuno-stimulatory /

MDP-hydrophilic Strong Th2 - - - Use in w/oMDP-lipophilic Strong Th1 - - - Use in o/w

Non-ionic block ? - / +++ +++ - Use in w/o or o/wcopolymers (CRL 1005)

Saponins Strong Th1, Th2 - - - Form ISCOMs, use withliposomes, MPL

Lipid A (MPL) Strong Th1 - - - Use with o/w, liposomes,saponins

Cytokines Various - - - Use with particulate adjuvants

Carbohydrate Mod Th1, IL-1 +++ - - Preferably conjugate?polymer

Characteristics of particulate adjuvantsAdjuvant Immunomodulation Targeting Presentation CTL Comments

Adapted from Cox and Coulter. 1997. Vaccine. 15: 248-256

57 :

Licensed adjuvants for human vaccinesAdjuvants aluminium salt, emulsion MF59, particulate virosome

microbial product (with particulate adjuvant)

Current licensed vaccine adjuvants in humans

Licensed Antigens CommentsAluminium salt DPT, Hib, HBV, HAV, Depot effect, APC

pneumococcus, antrax, uptake, Th2 skewcholera, rabies

Emulsion MF59 Influenza APC uptake

Particulate virosome HAV, Influenza APC uptake

Microbial product(with particulate adjuvant)

E.coli LT Influenza (withdrawn) BindMPL HBV, melanoma GangliosidesRibi-529 (synthetic lipid HBV TLR-2 and -4A mimetics)Chlorella endotoxin B Cholera TLR-2 and -4

Simon and Edelman, 2006

Side effects of adjuvants adjuvant

aluminium salt parenteral

aluminium salt adjuvant

Freunds Complete Adjuvant (FCA)

Freunds Incomplete Adjuvant (FIA) granuloma sickness behavior

Particulate Non-particulate ISCOMs side effect saponin ISCOMs

58 :

Aluminium salt parenteral usually not severecontact hypersensitivity, subcu. Noduleserythema, granulomatous imflammation

FCA, FIA parenteral abscesses, granulomaFever (FCA)autoimmune diseases, arthritis (FCA)possible carcinogenicity

LPS, MPL parenteral LPS: high toxicityMPL: low toxicity

MDP parenteral pyrogenicity, uveitis, arthristisCT, CTB mucosal Enterotoxicity (esp. human)Liposomes parenteral/(oral?) NoISCOMs, saponins oral/parenteral saponins: local reactions, hemolysis

ISCOMs: noMicroparticles oral/parenteral no

Side effects of adjuvants

Adapted from Horzinek et al. 1997. In: Veterinary Vaccinology. P 131-152.

Adjuvant Route of injection Side effects

Ideal adjuvant Adjuvant immediate, long term side effect minimized manufacturingvariation enhance protective immunity improve efficacy H5N1 adjuvant stability 2 , biodegradable , adjuvant

Final thoughts adjuvant

adjuvant 2 alum adjuvant adjuvant adjuvant HIV, Influenza protective mechanism memory cells adjuvant

59 :

antigen adjuvant

Antigen(s): 1, 2 3 7

Adjuvant: adjuvant 1, 2 3

Acid or base: pH peptide pH

Buffer: buffer adjuvant pH pH

Preservative: Thymerosol preservative

Purified water:

stabilizer gelatin antigen adjuvant aseptic technique sterileproduct

3 (intradermal), (subcutaneous), (intramuscular)

2.3 Vaccine delivery system...

How Vaccine Work2.

60 :

langerhanscell immuneresponse monocyte

1.

2. 1

3. WHO 5

4. syringe

5. hydrolysis

6.

7.

single dose

, syringe,

. . . . .vaccine delivery system

Needle free vaccine delivery system Thermostable vaccines and vaccine

vial monitors Auto-disable (AD) syringes and

safety boxes Monodose prefilled injection devices Point-of-use sharps processing

61 :

Needle free vaccinedelivery system Thermostable vaccines

Needle free vaccine deliverysystem

3

1. Needle free injection devices :

2. Transcutaneous delivery systems : (topicalproduct)

3. Mucosal delivery systems:

1 2 Needle free injection devices

Jet injectors device

1940 (nozzle) intradermal, subcutaneous intramuscular

device

62 :

Needle free injection devices 3

1. Multiple-use nozzle jet injectors 50 body fluids single dose jet injectors

2. Disposable cartridge jet injectors(single-use nozzle devices) single dosejet injectors Biojector Bioject Medical Technologies

InjexTM Equidyne Systems3. Powder injection

injector epidermis langerhans cell immune cells single dose auto-disable cartridges LectraJet HS

63 :

Transcutaneous delivery systems topical product

langerhans cell lymph node langerhans cell lymphocyte

Transcutaneousdelivery systems

a. (Hairfollicle) stratumcorneum ( ) DNA

b. epidermis (tape stripping)

c. (micropore)

radio wave (porous) epidermis

d. colloidal carrier topical product (hydrophobic) epidermis

e. ultrasound stratum corneum epidermis

f. adjuvant (adjuvant patch) (surfactant) stratum corneum

64 :

g. (electroporation) 10 - 20 mV

h. (microneedle) epidermis dermis nerve cells

Microneedle : silicone biopolymer 25-1,000 epidermis dermis

Silicone : stratumcorneum

stratum corneum biopolymer microneedle amylopectin, CMC polymer epidermis dermis

topical vaccine SLN: Solid Lipid Nanoparticles surfactant large scale production DNA vaccine plasmid DNA(pHIS-HIV-hugag) .. ()DNA SLN lipid surfactant form complex plasmid DNA SLN

65 :

SLN-pHIS-HIV hugag complex, CS-pHIS-HIV hugag complex Naked-pHIS-HIVhugag intradermal topical IgG titer

66 :

SLN Chitosan Naked pDNA intradermal topical topical SLN topical chitosan topical naked DNA SLN-pHIS-HIV hugag complex CS-pHIS-HIV hugag complex label IT TM-Rodamine labeling Kit pDNA 3 Confocal microscope SLN stratum corneum Chitosan Naked pDNA Mucosal delivery systems

mucous membrane Mucosa-associated lymphoid tissue;MALT (Nasal-associated lymphoidtissue; NALT), (Bronchus-associatedlymphoid tissue; BALT) (Gut-associated lymphoid tissue; GALT) , vaginal rectum mucous membrane

MALT microfold

(M) cel ls , antigen presenting cel ls(macrophages and dendritic cells), CD4+ CD8+, T-cells B-cells secretory immunoglobulin A (s-IgA) systemic immuneresponses (serum IgG production,lymphocyte prol i feration, cytokineproduction cytotoxic lymphocyteactivity)

Mucosal delivery systems Oral, Nasal Aerosol Vaccine

* Oral Vaccine

1. Trivalent live attenuated Sabin oralpoliovirus vaccine (OPV)

2. Oral live attenuated Salmonella typhiTy21 vaccine

3. -Oral live cholera vaccine strain CVD103-HgR-Oral monovalent (anti-O1)recombinant B subunit killed wholecell vaccine-Sweden-Oral killed whole cells bivalent(anti-01 and V. cholerae 0139)vaccine-Vietnam Gut-associated

lymphoid tissue (GALT)

67 :

Lamina propria plasma cells,macrophages, neutrophils ,eosinophils mast cells

Intraepithelial lymphocytes ( epithelial cells mucosalmembrane)

Isolated lymphoid follicles (intestine colon)

Peyers patches ( lymphoidfollicles ) Peyers patches B lymphocyte 40-70 T-cell 10-40

specialized cell M cells follicle-associated epithelial (FAE)

normal epithelium lymphoid tissue secretoryIgA effector T cells

polymer polymer PLGA WHO (USFDA) PLGA nanoparticle

68 :

5-10 Peyerspatches GALT mucosal 5 mesenteric lymphnode polymer synthetic polymer Polyalkylcyanoacrylate particles (100 nm) Polyacrylamide particle (2.55 mcm)

oral polymericvaccine polymer PLG, Alginate, Starch,Proteinoid, CAP ( synthetic polymer)

liposome liposome adjuvant

oral vaccine JE antigen JE virus Chitosannanoparticle Chitosan biopolymer Ionic gelation Emulsion coacervation 100-200 nm

69 :

hydrophilic 60 Caco-2 cells GI tract Raji cells M cells nanoparticle FACS spectra

JE solution mean 1 IgG titer JE oralsolution

mean JE solution

* Nasal Vaccine

USFDA Flumist (live cold-adaptedtrivalent influenza vaccine)

10-30

70 :

5 10-30

AdvanceDrug Delivery Review challengeinfluenza virus 3 Intranasal vaccine(IN), Intramuscular vaccine (IM) Controlgroup influenza virus IN IM Control group

IN Tetanustoxiod antigen nanoparticle 10 30 IgG antibody level solution IgG titer 24 IgGtiter 24 nanoparticle

polymer

muco adhesive polymer muco adhesive epithelialcell

* Aerosol Vaccine

5 Nebulizer ( ) Diskhaler Air Jet nebulizer

Aerosol vaccine Clinical trial Measles vaccine

From Csaba, et al., ADV. Drug Delivery Review, 61, 140, 2009

71 :

3 Jet injector, Transcutaneousimmunization Mucosal immunization

* Thermostable vaccines

(Needle free vaccinationdelivery systems)

hydrolysis hydrolysis freeze dry spray dry stabilizer cryoprotectant stabilizer

Needle-freeJet injectors

Transcutaneousimmunization

Mucosalimmunization

Multi-use-nozzlejet injectors

Disposable-cartridgejet injectors

Powder injectors

Vaccine and adjuvantpatches,irnnunostimalantpatches, andmicroneedles

Oral vaccination

Nasal vaccination

Aerosol vaccination

Speed of vaccination

No occupational risk(and low patient topatient risk) ofblood-borne pathogentransmissionParenteral vaccinedeliveryNo occupational orpatient to patient riskof blood-borne pathogentransmissionSpeed (especially withhigh workload varieties)Cold chain not neecled

Ease of delivery

No painNo risk of occupationalor patient to patientblood-bone pathogentransmissionExperience with use oforal polio vaccine

Ease and speed ofdelivery

No risk of occupationalor patient to patientrblood-borne pathogentransmissionNo pain

With edible vaccines :No cold chain, low costEase of deliveryNo risk of occupationalor patient to patentblood-borne pathogetransmissionNo painEase of deliveryNo pain

Pain similar to needleand syringeRare blood-bornpathogen transmission

Pain sirnilar to needleand ssyringe

Need for standardizationof cartridgesNeed more data re : safetyand efficacyRequires use of anadjuvant to stimulateimmune system

Rare vaccine-associatedparalytic poliomyelitis(VAPP)Rare reversion of oralpolio vaccine virus towild typeRotavirus vaccine andpossible link tointussusception

Bells palsy withinactivated influenzavaccine (with I.Tadjuvant)

Need more data re : lackof disease transmissionbetween subjects

Human SubcutaneousInjector (H15-500), withprotector cap

Biojector2000

Injex TMLectraletPowderject

Patches, such as thoseby IOMAIMicroneedles, such asMicro-TransTM

Oral plio vaccine

Oral typhoid (Ty2la)vaccine

Oral cholera(CVD 103-HgR)vaccine

Live attenuated bacteriaas vectors (expressingforeign antigens)Transgenic plant ediblevaccinesLive cold-aclaptedtrivaleent influenzavaccine(FluMistTM)

Live attenuated measlesvaccine

Advantages Disadvantages Examples

72 :

sugar trehalose freeze dry lactose spray dry amorphous form

stabilizer potency

purified water Autoreconstitution device diluent purified water

73 :

gene cell therapy

2511 H3N2

BCG

3.1 Researcher perspective in vaccinedevelopment

..

3.

74 :

1. 2. 3. R & D management4. 5.

3 1 Achillestendon ( : single or a few protective antigenvirulence factor) toxin

- host filamentous hemagglutinin pertussis

toxin

- host HA influenza virus block hemagglutinin

toxin , toxin toxin toxoid

toxin microvilli toxin 50-60 1 toxin diffuse 50 nanometres toxoid IgG IgA colonize

(scarlet fever) toxin

75 :

(pertussis) toxin pertussis toxin 4-5 2

- polysaccharide glycolipid

-

- extracellular intracellular cellular immunity

- serotype 2-3 serotypes

1

2 completeimmunity serotype

Pathogens serotype 1. Influenza: trivalent seasonal vaccines2. Polioviruses 3 serotypes3. Human papillomavirus : quadrivalent

vaccines

4. Streptococcus pneumoniae >90serotypes (vaccines available for 23serotypes)

5. Neisseria meningitidis6. Dengue viruses 4 serotypes7. Leptospira interrogans >200 serovars8. Streptococcus pyogenes >100

serotypes antibody rheumatic fever

protective antigen polysaccharide T cell independent

protective antigen polysaccharide polysaccharide Hib

Polysaccharide antigen (T-independentantigen) B cell IgM

T-dependent Ag

lg GB cellsTh cells

P rotein P R P

ILs

76 :

T-independent B cell IgG protein conjugates

polysaccharide

- Haemophilus influenzae : serotypea-f, type b (Hib) protective antigen PRRP (not immunogenic) protein conjugation

- S. pneumoniae : proteinconjugation

- N. meningitidis : proteinconjugation

- Salmonella typhi: Vi capsule proteinconjugates clinical trials

- E.col i : O157:H7 O-specif icpolysaccharides : protein conjugates 3 conjugate vaccine

definiteprotective antigen Leptospira, Neisseriagonorrhea, TB, Burkholderia, other bacteria,fungi, protozoa. Intracellular pathogen salmonella,TB, Shigella

cell mediated immunity T cell macrophage

Intracellular - Salmonella typhi : live-attenuated

strain or Vi antigen- Mycobacterium tuberculosis : BCG- Shigella : 4 species, 37 serotypes,

Shiga toxin Direct cell-cell invasion cell mediated immunity Shigella cytoplasm cytoplasm host

inactivatedvaccine

3 complete immunity Staphylococcus aureus, malaria, HIV

77 :

Helicobacter pylori (clinical trials Phase 3) TB, HIV,leprosy

(Vaccine Developments)

discovery, pre-clinical testing clinical trials

clinical trials phase 3 gold standard of efficacy phase 1 phase 2 phase 3 immunological markers for efficacy inphase 3 correlation marker marker phase 3 trials

phase 3 neutral izing correlation protection phase 2 neutralizing antibody complete phase 3

trial marker phase 3 TB marker correlate phase 3 phase3 5 marker AIDS, malaria, TB, S. aureus, H. pylori

clinical trial

immunological marker phase 1 2 neutralizing antibody, animalmodel develop animal model TB (granuloma)

(In vitro testing) in vitro testing

78 :

in vitro testing pathophysiology guideline

Management of Vaccine R&D- Stakeholders- Financing- Stage-Gate protocols

Stakeholders 1. 2.

financial

3. Product development manager , .,

4.

Financing

Stage-Gate protocols

protocol Stage-Gate protocols FDA

Gate Keeper product stage stage

79 :

Dengue virus

4 4 serotypes Dengue virus

Dengue virus

: Dengue

virus 2 50-60

(envelope) : E

180 90 E E E E Dengue virus

(nucleocapsid) : C RNA 1

E E

3 - :

- :

3.2 Preclinical vacine development - Dengue vaccine

..

3.

80 :

- :

E serotype E serotype ()

..... Dengue virus

RNA 1 10 10 3 7 3 E, PrM, Capsid () 7 precursor protein

81 :

3 Interferon 2A, 4A, 4B

interferon

immature mature (immature) immature mature Maturation

immature

82 :

mature

(immature) pH dengue

endoplasmic reticulum vesicular transportpathway

host

cell monocyte, macrophage, dendritic cell identified

116 42

Dengue virus Flavivirus JEV (Japaneseencephalitis virus) WNV (West Nile virus) (Yellow fever; YEV)

83 :

Dengue virus

4 serotypes DENV-1,DENV-2, DENV-3 DENV-4 serotype 40 4 serotypes serotype (subtype/genotype) DENV-1 DENV-4 3 genotypes DENV-2 DENV-3 5 genotypes 10-15

(strain)

4 serotypes subtype

Dengue virus monocyte, macrophage antigenpresenting cell (innateimmunity) (adaptiveimmunity) E Neutralization

E E monocyte macrophage NS1

84 :

E T lymphocyte neutralized T lymphocyte

40 2-3

serotype () serotype

(3-6 ) serotype 4 serotypes

Flavivirus Flavivirus

(Yellow fevervaccine)

(Japaneseencephalitis vaccine) SA-14-14-2

(Japaneseencephalitis vaccine) SA-14-14-2

Dengue virus

85 :

1. (Whole, Killedvaccine)

2. (Live,attenuated vaccine)

3.

( 37 )

4 serotypes serotype

2

1. 2 Sanofi Pasteur Walter ReedArmy Institute of Research GlaxoSmithKline

2. Acambis Sanofi Pasteur US-NIH, US-CDC, US-FDA

US-NIH DENV-1, DENV-2, DENV-3 DENV-4 4 Acambis Sanofi Pasteur US-NIH, US-CDC US-FDA 3 Sanofi Pasteur

86 :

neutralizingantibody (P, PrM) DENV-1, DENV-2, DENV-3 DENV-4 US-NIH DENV-1, DENV-2, DENV-3 DENV-4

2

-

- , -

(neutralizing antibody)

-

-

1. DNA vaccine prM+E ( ) (iDNA : FL cDNAclone with pseudolethal mutations)

2. Subunit vaccine E (Recombinant E protein) (prM+E subviral particle)

3. Pseudo-infectious virion

4. Other vectors

87 :

Dengue vaccine Influenza vaccine 2 Dengue vaccine immuneresponse Influenza vaccine pandemic

Structure of virus particle Influenza virus

2 haemagglutinin (HA) neuraminidase (NA) antibody response CTL (cytotoxic T lymphocyte)

antibody response haemagglutinin segment strain reassortant segment reassortment H1N1 reassortment

Antigen variation Antigenic

shift Antigenic change 2 Influenza

Antigenic drift mutation drive immune response host population

Antigenic shift HA number type H1, H2,H3 H5 H1N1 Antigenic shift

3.2 Preclinical vacine development - Influenza vaccine

..

3.

88 :

H1N1 2009 H1 H1 100 2461 H1 antigenicity immunology immune response H1N1 2009 crossingantibody

antigenic evolution mutation epitope HA mutation epitope antibody microneutralization haemagglutination inhibition influenza titer HItiter H3N2 Influenza virus map antigenic cartography

89 :

seasonal influenza 2-3 mutate immune response host strain antigenic epitope ( Antiqenic cartoqraphy 2 ..) recycling epitope concept H3N2 H1N1 seasonal influenza genetic map antigenic map strain strain genetic sequence ( antigenic study serum strain titer strain standard serum ) sequence genetic distance antigenic distance (drift) random immune response host H1 H3 antigenic epitope receptor

binding site H3 a, b, c, d epitope map amino acid H3N3 mutation a antibody epitope b, c, d epitope epitope (probability) epitope sequential epitope epitope bias bias target epitope epitope epitope antibody antibody epitope antibody conservedepitope antibody

90 :

pandemic split vaccine subunit vaccine purification pre-clinic 2 recombinanttechnology cell culture antigenic diversity conservedepitope haemagglutinin (HA) neuraminidase (NA)

whole inactivated

vaccine scale up cell culture whole virus vaccine (reactogenicity) reactogenicity immunogenicity H5 whole virus vaccine

Split lipid envelope

Subunit purificationprocess haemagglutinin neuraminidase

influenza vaccine reassortant strain strain antigen strain virus strain

1. 2.

influenza vaccine purified centrifugation process 2 reassortment reassortant antibody suppress donor internal gene 8 Peurtorigo 8/64 reverse genetic technology

91 :

live

attenuated vaccine ( intranasalvaccine) antibody pre-pandemic reassortment pandemic Live attenuatedvaccine 30 pandemic killed vaccine scaleup 10 switch killed vaccine live attenuated vaccine antigen

Annual Influenza vaccine productiontimeline

Time line seasonalinfluenza strain selection vaccine strain

purification testing antigenicity purity filling packaging

seedvaccine 1-2 1 monovalent production 4

regulator () pilot scale facility pilot production GMP safety side effect licensing Swine influenza vaccine side effect pandemic

92 :

93 :

influenza immunogenicity safety

H5 H1 seasonalinfluenza H5 H5 trial 90 seasonalinfluenza 15 haemagglutininpandemic H5

seasonal influenza immunogenicity H5 naive 2 influenza immunology crossing H1 H5 2 H5 (antigensparing) adjuvant immun-ogenicity antigen adjuvant Alum H5

94 :

clinical trial H5 multicenter trial adjuvant antigen sparing cross antibody H1 wave 2 3 wave 2, 3 antigenic drift antigenic drift 2-3 influenza adjuvant H5 intradermal rabies vaccine intradermal antibody response intramuscular 5-10 intradermal

..

- Influenza virus antigenic shift

- Influenza vaccine peak 2 Hepatitis B vaccine plasma vaccine seasonal vaccine 30 H1N1 increase seasonal vaccine 2 seasonal vaccine

. H5N1 reverse

genetic provide candidate vaccine H5N1 cell culture 2549 vaccine prototype US CDC UK NIBSC(National Institute for Biological Standards

95 :

and Control) (reversegenetic) 1194 1203 killed vaccine upstream process bioreactor culture cell vero cell downstream process culture animal testing,immunogenicity

bioreactor room bioreactor 4-5 animal cellculture microbial cell culture (yeast E.coli) pathogenic biosafety level 2

microcarrier cell

H5N1reverse genetic HA lot haemagglutinin antigen vero crell downstream bioprocess harvest, pre-filtration, cross-flowfiltration, gradient ultracentrifugation column chromatography

yield reverse genetic H5N1 vero cell 108-1011 pfu/ml titer

haemagglutinin 1:160 1:1,280 haemagglutinin protein 3-20 /ml culture haemagglutinin haemagglutinin HAprotein quantitate batch haemagglutinin fermentor reversegenetic 100 haemagglutinin 100 1 1 batch 300,0002,000,000 HA 100 downstreamprocess 50% recovery haemagglutinin 3 100 50,000300,000 1 100

2 serum homologous virus neutralized vary haemagglutinin 1, 3 7 adjuvant, mouse antiserum haemagglutination inhibition microneutral ization plaqueneutralization

3 2 strains (1203 1194) antiserum

96 :

inhibit inhibit titer 1:1,280 saline

homologous virus haemagglutinationInhibition 1:80 1:1,280 microneutralization titer 1:1,280

antibody 2 1194 2 3 2 titer neutralized 1:160 strain 1203 3

cross-neutralization 5strains 4 strains clade 1 strain A/Lao-Thai/286/2007 clade2 serum cell culture process influenza vaccine strain titer 1:40 strain

2 1:40 strain 3( 2006) titer 1:320 titer 1:40 1:80 positive control homologous 1:640 1:1,280 cell culture process antibody facility prototype US CDC UK NIBSC neutralized 5 clade antiserum 5 neutralized reverse genetic survivor 2 2 samples 2008 survivor 3 2 contact person antiserum neutralized reversegenetic antiserum neutralized

97 :

AIDS vaccine 2

1.

2. (infectedwith no disease)

3. slow diseaseprogression

HIV Neutralizingantibody, Cytotoxic T cells Helper T cell

HIV Neutralizing antibody(Nab)

Cytotoxic T cell (CTL) Helper T cell (Th) B cell HIV Neutralization CTL 10 T cell-based vaccine Neutralizing antibody

T cell-basedvaccine STEPtrials Phase 2b adenovirusserotype 5 (Ad5) (vector) 3 (Gag/Pol/Nef) trivalent vaccine T cell response ELISpot assay HIV protein preexisting antibody

3.2 Preclinical vacine development - HIV vaccine

..

3.

98 :

placebo Ad5virus (titer > 200) preexisting Ad5immunity

AIDS vaccine

HIV

- protectiveimmunity neutralizing titer , epitope T cell

- -

broad spectrum neutralizingantibody

- T cell-based vaccine protective immunity

- Later Phase (2b, 3) heterogeneous population sample size

animalmodel inoculum genitalmucosa (vaginal canal) 2 regional lymph nodes neutralizing antibody mucosal immunity systemic disseminated 2

(CTL+Nab) STEP trials neutralize HIV envelop coreceptor CD4 CCR5

Tomeras acuteinfection 100 copy sensitivity 50-100 copy 8 immune complex antibody anti-HIV anti-human surface molecule cellmembrane 2 gp41Ab 1 gp120 Ab 1 antibody non-functioning antibody neutralize control viremia

99 :

type specific antibody broad reactive

John Mascola Group broad reactive Ab 1 5 HIV positive broad reactive neutralizing titer

Johnson neutralizing Ab genetherapy antibody passive immunization passive immunization

vector recombinant technology adeno like virus vector (rAAV) genedelivery single doseIM monoclonalantibody gene monoclonal monoclonal antibody neutralize HIV SIV model (Simian immunodeficiencyvirus) 3

broadneutralizing antibody human monoclonalneutralizing antibody designneutralizing antibody based vaccine

design Ab-based vaccine design poorimmunogenicity glycan window of protection 2

HIV Vaccine

Preclinical AIDS vaccine development1. CTL epitope mapping2. HLA typing and epitope mapping

CTP epitopemapping HIV positive 250 , HIVnegative 150 Nab HLA typing central lab

CTLresponse HIV-1 AE peptide ELISpotassay population recognized Gag, Pol,Nef 80

30 epitopes recognized HIV new epitope HLA class I CO1O2IY9 selected pressure CTL 30 mutation 2 4

CTL-based vaccineCTL based vaccine clinical

trials

100 :

- Adenoviral replicons: adenovirus T cell protection preexistingantibody

- Poxcirus vectors (MVA, Canarypox,modified vaccinia, Fowlpox)

- DNA vaccine: adenovirus DNAvaccine active paper

David Weiner DNAvaccine Vaccine Research Center (Phase 1 safety andimmunogenicity evaluation of a multicladeHIV-1 DNA candidate vaccine) DNA vaccine needlefree IM injection antibody Western blotELISA 80 ELISpot prime-boost DNA primed boost adenovector T cell response specific HIV Gamma-Interferon

DNA vaccine DNA vaccine stand alone magnitude

, adjuvant delivery DNA vaccine needle freeinjection in vivo electroporation targeting dendritic cells

* DNA vaccine delivery systemNeedle free biojector :

In vivo electroporation :

Phase 1, 2 trials Cancer vaccine

* Preclinical development (HIVDNA vaccine R&D)

Thai BIOTEC GrantedProgram 2005-2010 HIV DNAvaccine Allergy vaccine HIVvaccine CTL epitope (Gag,Tat, Nef) Gag Codon optimization humanize (Gag, Tat, Nef,Pol) result Gag route of immunization high dose Toll-like receptor ligands CpG motif enhance cellular uptake

immunofluorescent staining anti-gag antibody protein expression humanized HIV-1 Gag DNA vaccine

101 :

needle free injector IM DNA needle free injector trauma injury local adjuvant cytokines, chemokines antigen presentation process

ID, IM needlefree immunized IM ID

DNA vaccine global HIV diversity (A, C, AG) South East Asia AE,B, C South America B, BF, F cladespecific vaccine development

Mosaic protein design to increasemore global coverage

Four mosaic proteins perfectly matched- 74% of 9-amino-acid potential

epitopes in global Gag sequences- 87% of potential epitopes matched

at least 8 of 9 positions. In contrast,a single natural Gag protein covered

- Only 37% (9 of 9 mers) and 67% (8of 9 mers)

Asian mosaic CRF01 (AE) Asian B mosaic random 100 base (randomrecombinant)

NCI plasmid expression 2 constructs non-mosaic mosaic antibody response HIV DNAplasmid vaccine mosaic non-mosaic mosaic DNA antibody response100% pHIS HIV Gag ELISpot mosaic DNA

prime-boost DNAprimed vaccinia boosted DNA alone AE peptide( mosaic mosaic AE, B computerized) DNA alone ELISpot 1000 spot plasmid prime-boost 1,700 spot (p=0.0087) peptideB prime-boost recognized B recognized AE intradermal needle freeinjection needle free spot (p=0.0043)

mosaic HIV-1 AE/B gag B immunogenicity AE mosaic gag

102 :

vaccinia B needle free injection

HIV vaccinedevelopment

Back to basic- identify immune

protection - broad neutralizing Abs

design antibody-based vaccine- improve T cell-based vaccine

Exploring new approaches- gene delivery neutralizing

antibodyClinical evaluation- Early phase trials

phase 2b homogenouspopulation

.

HIV

100 HIV vaccine 2526 26

2551 2 Montagneir Sinoussi HIV

HIV vaccine sterilizing immunity neutralizing antibody neutralize CTL Tcell-based vaccine vaccine neutralizingantibody CTL

HIV - HIV retrovirus

integrate host cell 2 1 gut-associated lymphoidtissue window of opportunity to preventHIV infection antibody neutralizing antibody mucosal antibody innate immunity

103 :

- HIV CD4 (CD4) immuneresponse

- HIV hypervariable mutation rate recombinant form HIV 9 subtypes, 34 CRFs (circulatingrecombinant forms) 150 URFs(unique recombinant forms)

- HIV CMI HMI high mutation rate envelop epitopes neutralizingepitopes sugar structure gp120 antibody neutralize

HIV Preclinical

broadly neutralizing antibodies challenge SHIV(HIV-like virus) live attenuated SIV

Clinical observation slow

progress delay progression HIV subgroup elite controllers viral load detect ( 50 copy) 20

exposed

HIV HIV 3 waves Wave 1 : concept neutralizing antibodies phase 1, 2, 3 recombinant gp120 neutralize primary HIVisolates Wave 2 : concept CMI CMI Wave 3 : wave

Study step

viral load T cell-based delayprogression Ad5 vector phase 2b proofof concept 2 concepts concept pathogenesis HIV mucosal immunity

104 :

HIV innate responses immunological assay evaluate vaccine

- neutralizing

antibody neutralizing antibody envelope CMI elite controller expand cohort el ite

controller immune mechanism

- support innovation

- clinic preclinic clinicaltrial animal model

-

105 :

Pre-clinical study

Pre-clinic

1. (pharmacology)

(Immunogenicity)

Pharma-codynamic studies of adjuvant

2. (Pharmacokinetics)

3.3 Safety and toxicology assessmentof vaccine in pre-clinical study

..

3.

106 :

adjuvant pharmaceuticaldoses forms

(Safety Assessment)

3. (Toxicology)

species (Immune response) species immune response immune response

parameter

(responsecomplex) animalmodel

107 :

immune response animalmodel immune response binding antibody targeted pathogen

EU (EuropeanGuideline) 1specie InternationalConference on Harmonization (ICH guideline) 2 species single dose study (clinical trial)

1.

(repeated dose toxicity study) :

vaccine regimen adjuvant

(dose levels) 2 immune response

Parameters single dose repeated doses local tolerance repeateddoses toxicity

introduce (cytokine production)

108 :

(Immune complex Assay)

- -

-

(Genotoxicity) (carcinogenicity)

- (Reproductive toxicity studies)

Rabies vaccine Influenza vaccine expose

2. (Reproductive toxicology studies) :

vaginal smear hot female smear sperm sperm

antibody antibody (IgG)

(Immunotoxicology)

109 :

(repeated doses) (adverse effects) (hypersensitivity) (autoimmune reactions) animal model (Systemic anaphylactic models) adjuvant

responsive genetic background false positive results antihistamine

(Autoimmunity)

Autoimmune disease immune protein

vaccine, antigen hostprotein autoimmune response

(autoimmune disorder) animal model autoimmune disease biological markers markers autoimmunity hypersensitivity reactions

(Live attenuatedvaccines) (adjuvant,stabilizer, additive)

(Intrinsictoxicity)

110 :

intrinsic toxicity adjuvant adjuvant cross-activity antigen (combination) immune response

biologicalprocess pathway

host cellcontamination protein celllines human cell lines

(species

specificity) specie

(Non-clinical Assessment)

non-clinical methods model

(non-clinicalsafety)

111 :

(therapeutic dose) therapeuticdose

pre-clinical study pre-clinical study non-clinic non-clinic Good Laboratory Practice (GLP) product literature non-clinical safety, protectionaspects clinical study

(Environmental Risk Assessment)

(Genetically Modified Organisms-GMO) host cell introduce genome DNA host DNA

(Cross-references)

112 :

113 :

(soundscientific rationale) risk benefit ratio (Animal study) (Immunogenicity) (GoodManufacturing Practice- GMP)

, pre-clinic clinic 1 immunogenicity 2 3 (Efficacy)

1. (targetpopulation) (Inclusion criteria) (Exclusioncriteria)

3.4 Clinical trial for candidate vaccine :Clinical evaluation

..

3.

114 :

2. (Outcomemeasurement) (Primary endpoint) endpoint

3. Safety, immunogenicity, efficacy4.

outcome endpoint pre-cancerouscondition surrogate marker Hepatitis B bridging study

5.

6. 7.

Superiority trials Non-inferiority trials (one-side equivalence) Two- side equivalence trials

8. 9.

phase 1 (Last dose ofvaccination)

10.

(Clinical Study) 4

1

() dose response optimal dose 1 log maximal safety dose log

115 :

guideline 1 , , multicenter

(live attenuatedvaccine) (Shedding) (Transmission) (Genetic variability) 1 2 Cholera vaccine

2

2 2

multiplesites optimal dose 3 milestone criteria 2 milestone 3 milestone criteria

(Efficacy trial) 2 optimal immune response

2b (Test of concept study) proof of concept 3 2b (Preliminaryassessment) (Phase 3 study) 2b

Virus-like particle test ofconcept monovalent 2 (GSK Merck) 3

3

(Efficacy) double blind controlled study

116 :

1 2 placebo active multi-centre endpoint (correlate of protection) side effect

clinical intervention 5

3 protocol

4 Post-marketing study

(Safety surveillance) head to head comparison

outcome, new dosage newformulation

Bridging studies scenario endpoint bridging immune response bridgingstudies

bridging studies EU . . bridging study 3

bridging studies new dosing schedules immuneresponse 9-10 3 16-26 immuno-bridging studies immune response 9-26

2

117 :

match 2-3 10 20 25-26

good clinicalpractice , ,, , , ()

, ,

standardizedmethods

case record form Varicella vaccine 42 Hepatitis B 20 (16 ) Phase 3 1 Rota virus 10

118 :

119 :

endpoint (outcome) HIV endpoint endpoint

Primary endpoint : endpoint

Secondary Endpoint : endpoint (risk behavior)

placebo

2 (post exposures) anti-cocaine anti-nicotine

(Smallpox) smallpox Edward Jenner Jenner

3.4 Clinical trial for candidate vaccine :Endpoints

.() .

3.

120 :

Endpoint endpoint

1 (immunogenicity) (Clinical benefit) Test of concept : (Efficacy) 3 30 60 30 HIV , viral load , Hard endpoint : clinicalendpoint immune responseendpoint immune correlate FDA animal rule endpoint hard endpoint

Soft endpoint : Clinical endpoint : sterile fluid clinical endpoint Correlation of protection : immune correlate 1 2 3 immune response immune response immunecorrelate Animal rule : endpoint

121 :

kinetics dynamic 1 side effect side effect animal rule 1 2 3 animal rule Anthrax, Botulism, Plague, Tularemia, Ebola Surrogate endpoint : clinically meaningful endpoint Surrogate endpoint CD4 200 microgram CD4 Surrogate endpoint Surrogate endpoint clinical trials HPV HPV cervical intraepithelial neoplasia 2 3 (CIN 2,3) Adenocarcinoma in

situ (AIS) 5 20 CIN 2, 3 AIS surrogate marker CIN2, 3 AIS

endpoint vascular event transient ischemic attack sensitivity

(therapeuticvaccine) cancerantigen cellular CD8 herceptin antibody immunotherapy

shedding endpoint Neurovirulence

122 :

123 :

4

(Regulatory& Guidelines)

Good Clinical Practice (GCP) GCP ICH guideline ICH guideline

GCP GCP CFR(code of federal regulation) (E) E6 GCP endpoint E6 E9

3.4 Clinical trial for candidate vaccine :Statistical issues

..

3.

124 :

primary endpoint

HIV (Infection rate) CD4 ICHguideline

E1

1. success rate control group

outcome outcome infection rate infection rate control 2,500 infection rate infection rate incidence SD outcome

2. effect size

IDU (Injecting Drug Users) 5.6 effect size HIV 30 50 effect size

125 :

3. Type I error

5%

4. Type II error Type I error

error 10% 20%

placebo 25% treatment 65% 65%

21 protocol

dropout rate lost to follow-uprate dropout rate

study profile

2

* (descriptive statistics) (inferential statistics)

- descriptive statistics

- inferential statistics

n = 7.849 x [ p1(1 - p1) + p2(1 - p2)](p1 - p2)

2

n = 7.849 x [ 0.25(1 0.25) + 0.65(1 0.65)](0.25 0.65) 2

n = 7.849 x 0.415 = 20.40.16

126 :

* 2 Parametric Non-parametric statistics

- Parametric statistics

- Non-parametric statistics distribution free statistics

1)

2) viral load (length of stay) parametric statistics

3) Central Limit Theorem non-parametric statistics

MannWhitney U-test, Kruskal Wallis test T-test ANOVA test outcome chi-squared test infection rate 2 incidence

repeatedmeasure baseline baseline 2, 3 4

outcome time to event(survival) outcome time to event 2 endpoint

protocol (Randomized control trials) 3

1. Intention-to-treat A ()

127 :

B () 1,200 5 6 outcome intention-to-treat

2. Per-protocol protocol 5 6

3. As treated

per-protocol intention-to-treat (inclusion and exclusion criteria) (missing data)

* Subgroup analysis

subgroup analysis sample size subgroup analysis protocol subgroup analysis

DSMB (Data and SafetyMonitoring Boards) DSMB USFDAguideline DMC (Datamonitoring committee) DSMB (IRB =Institutional Review Board) IRB protocol DSMB safety report IRB DSMB

128 :

DSMB DSMB

(InterimAnalysis) Z test type I error 5% 5% placebo +1.96 Z 1, 2, 3 placebo 4 placebo 1.96 5% 1 5% 5 5% p-value 1 5% 5

14.2% DSMB

Type I error Pocock +1.96 Haybittle, Peto Obrien &Fleming DSMA

Futility Analysis 3

129 :

1. 2. clinical trials

3

3 2549-2551 1-5

( 70,000-80,000 )

3

3,000 7 () 3-14

3.5 Candidate vaccines in clinical trials :Efficacy and safety of dengue vaccine :

Thailand phase 2b..

3.

130 :

3

7 3,000 28,000 5,000 3,400 572

Symptomatic Laboratory ConfirmedDengue Virus Infection 2006-2008 ELISA, IgM, IgG, PCR PCR positive biologyconfirmed virus culture 3 2549 51 , 2550 111 2551 156

2549-2551 2549 2550 2550 2551 2550

2549-2551 2549 1.94 3.4 5.5 2550 2551

Number of Febrile Episodes andSerologically Confirmed Dengue Casesby Month 2006-2008 PCR positive denguecases 12 (febrile episode)

Dengue Serotype Distribution 4 serotype 2549 Dengue 1 Dengue 4 2550 2551 Dengue1 Dengue 2 2551 Dengue 1 42 Dengue 2 31 serotype 1 2

3 vaccine trial

131 :

viremia 855 700 2,000 12

2551 Sanofi Pasteur phase 2B (Dengue Vaccine Trial: Efficacyand Safety of Dengue Vaccine in HealthyChildren Aged 4-11 Years in Thailand) 4,000 4-11 TetravalentChimeriVaxTM Dengue Vaccine yellow fever virus vaccine prM E

Dengue vaccine monovalent vaccine 2545 trivalent tetravalent 2548

CYD06 CYD05 design naive dengue antibody dengue virus 2551 2-11

1 2 3

1 control placebo Typhi millium control yellow fever control adversereactions Typhi millium local reaction severe local reactions Typhimillium dengue vaccines

132 :

systemic severe reaction denguevaccine Typhi millium

(neutralizingantibody) serotype 1 2 group 1 3 doses group 2 2 doses group 1 3doses serotype 1 3 90 serotype 2 100 1 serotype positivity serotype3 4 serotype 1 2

GMT (Geometric Mean Titer) serotype 1, 2, 3 4 1, 2 3 3 serotype 100 viremia 1 2 3 viremia PCR plaque viremia

chimericdengue vaccine 3

133 :

(dengue vaccine challenges)1.

4 5 (monovalent) 4 (tetravalent) 1

2. (pathophysiology)

3. animal model

4. (immune correlate of protection) endpoint

5. endpoint

6. (endemic area)

3.5 Candidate vaccines in clinical trials :Tetravalent live attenuated dengue vaccine

phase 2 updateDr.Stephen J. Thomas Armed Forces Research Institute of Medical Sciences

3.

134 :

WRAIR / GSK LAV product WRAIR/GSK

4 types DEN-1 (Pacific), DEN-2(Thai), DEN-3 (Thai) DEN-4 (Colombia) type serial passage PDK 27, 50, 20 6 (combination)

(pilot lots) 2535-2547 (non-human primate) (phase 1) (adults) 150 (unprimedchildren) 41

1

Dengue Vaccine Candidates 1

(phase IIB)

(phase II)

(phase I)

Mahidol University and Sanofi Pasture(MU/SP)

Walter Reed Army Institute of Research(WRAIR) and GlaxoSmithKline (GSK)

National Institutes of Health (NIH) andJohns Hopkins University (JHU)

Naval Medical Research Center (NMRC)

GenPhar

Human Biologicals Institute (HBI)

Walter Reed Army Institute of Research(WRAIR)

Inviragen ()

US FDA

live attenuated Chimerivax

Live attenuated (LAV)

delta 30 genetically engineeringmutation (monovalent)

DNA

Live attenuated (LAV)

Cad-Vax (complex adenovirusvector)

R80E

PIV

PDK53

mutF

135 :

85 (neutralizing antibody) 3 4 (trivalent or tetravalent)

2 2545 seed virus certified diploid

cell line PDK

2 2549 3 (TDEN-001, TDEN-002 TDEN-003) 2

2 live attenuated dengue virus vaccine 2 WRAIR/GSK

TDEN-001(.. 2549-2551)

TDEN-002(.. 2550-2551)

TDEN-003(.. 2550-)

18-45 88 ()

20-25 120 ()

12 -50 636 PuertoRico ()

- (solicited symptoms)

- (spontaneous adverse events)

- -

- - - 21 - diary card 30

2-14

136 :

TDEN-001 WRAIR Clinical Trials

Center 2 (0, 6 ) 2 placebo (vaccine A & vaccineB vs. placebo)

(ocular) flu-like syndrome A B (solicited symptoms) placebo A 1 36.4 1 77.3 B 42.9 81.0 placebo 33.3 38.4 (overlapping

of 95% confident interval = no significantdifference)

Neutralizing antibody response 7 (1 2) (unprimed) (>cut-off) DEN1,2, 3 4 A 73, 80,67, 67 B 83, 100, 83, 73 placebo 0, 0, 0, 0 () 1 2 3 4 type (>cut-off) A 78 B 86 placebo 5

- Dengue viremia determination

- - Myocarditis

qRT-PCR serotype-specific (sensitive/reproducible/high throughput)- First generation- Second generation- Limitation of detection (LOD)

DENV-1 : 12.5 PFU/ml DENV-2 : 25 PFU/mlDENV-3 : 12.5 PFU/ml DENV-4 : 50 PFU/ml

(immunogenicity)- - Anti-DEN antibody micro-neutralization assay- CD4+ T-cell response intracellular cytokine staining- B cell memory ELISPOT

137 :

Viremia 3Follow up 1 : 2, 5, 8 12 Follow up 2 : 5, 8, 12 14 Follow up 3 : 30

viremia > LOD 1 A 12 1 DENV-4: 4.9 x 104 GEQ/ml ( 485PFU/ml)

2 3 (booster) 21 A B 9 12 2 5-12 1 3 2

placebo 2

- neutralizing antibody 4 type

- 1 moderate to highimmunogenicity 2 type

- 1 strain B

- CD4+ T-cell memory B-cell

TDEN-002 AFRIMs/

20-25 2 (0, 6 ) 2 (vaccineA and vaccine B vs. placebo)

dengue micro-neutralizationassay 3 A, B placebo multivalent dengue antibody> 1/10 77.5, 85.0 87.5 83.3 JEPRNT > 1/10 34

A 1 50.0 51.3 B 41.0, 31.4 placebo 32.5 23.1

138 :

A > B > placebo 1 2 A 1 87.5 71.8 B 69.2, 54.3 placebo 65.0 53.8 A > B = placebo 1 2

3 (grade 3 adverseevent) 3 A 3 21 1 1 2 2 1 21 1

( + viremia) 80 157 (unsolicitedadverse event) 2

4 (tetravalent) 67, 77 85 A, B placebo (tetravalent) 97 100 placebo 87

viremia > LOD 1 placebo (713 PFU/ml) A 3 B 2 5

viremia A viremia DEN2 2 1 2 16 9 PFU/ml B 1 DEN2 DEN3 1 2,343 51 PFU/ml

2 WRAIR/GSK LAV product1. 2. (immunogenicity)

o TDEN-001 (unprimed, US adults)* Dose 1-immunogenic/Dose 2-

brodens response*

(naive) type (monovalent responder)

o TDEN-002 (primed, Thai adults)* Single dose broadens immune response* Baseline monovalent status

broadened to tri- or tetra-valent

o 2 (A B) 1

* TDEN-003 power ()

* interference

139 :

TDEN-003 o

* *

o power A B* viral interference concept

* (manipulation of viral concentration)

*

o *

(specific candidate)

o TDEN strain transmissibilityo Continue TDEN process development

140 :

141 :

27 2524

2 HIV

2550

(UNAIDS) 33 2.7 2 ( 22 , 1.9 ) ( 4.2 , 330,000 )

1 2.5

HIV HIV

(Intravenous DrugUsers: IDU) (Sex worker) (Client)

3.5 Candidate vaccines in clinical trials :HIV vaccine

. () 3

3.

142 :

2531 , 2533 2538-2553

2570 HIV 5 2

:

( Dr.Bonnie Mathieson) :, , , , , , , ,

3 UNAIDS :,

2533-2534 100,000

HIV

...... HIV

HIV

143 :

HIV HIV strainsubtype HIV mucosal blood borne host cell DNA

2550 3-9 8-20 2558

1. Basic research :

2. Targeted research (Preclinical and

clinical) : reagents 1, 2 3

3. Product Development :

3

1

144 :

() 2

() 3

3

2536 , , ,

2537

10 trials 1 1 2 2542 3 2 trials

3 1 2 ALVAC vCP1521 AIDSVAXrGP120 B/E (prime-boost) ALVAC AIDSVAX source subtype E

: prime boost HIV

145 :

viral load set point : Community-

based, Randomized, Double-blind, Placebocontrolled 1:1 16,000 18-30

: 0, 1, 3, 6 0 1 prime vaccine ALVAC 3 6 prime boostvaccine ALVAC AIDSVAX 6 3 HIV

: 2 4

: 40 Screening sites, 7 1 Clinical sites

Screening sites Screening sites 5-7

29 2546 20 2546 30 2548 Screening sites 47 60,000 26,675 1 16,402 31 2549 4 13,978 26 2552 3 1 16,402 4 13,978 2552 6

146 :

90

(PotentialBenefit of Vaccines)

2 (Individual benefit) (Public benefit)

HIV HIV

E B, C A

2536

3 2 trials

prime-boost ALVAC AIDSVAX 3

- Adenovirus type 5 ( ) 1/2

- MVA 1

- DNA+Fowl pox 1

1 DNA+tientan replicable

3 3 1

147 :

HIV 2546 3 2548 T-cell vaccine

3 1. .. 2527-2546 : Antibody inducing

vaccine 2546 trials

2. .. 2538-2553 : T-cell inducingvaccine Adenovirus Merck DNA+Adenovirus type 5

3. .. 2543-2558 : membrane vaccine T-cell vaccine trial

- Preclinical

Clinical Phase 1 27

- Clinical Phase 1 Phase2 17

- Phase 2 Phase 3 16 - Phase 3 5

1. animal model, immune response

2. 3.

4.

27

148 :

149 :

2464,2500 2511 15

General & Rationale of vaccination 2549

50 2553 75 ( 65 ) 2543 60 90 2553

300,000-700,000 /

Antigenic drift & shift

neuraminidase hemagglutinin 8 HIV HIV Antigenic shift Antigenic drift

Antigenic drift : neuraminidase hemagglutinin

Antigenic shift : 8

3.6 Influenza vaccine. .

3.

150 :

multiply receptor antigenic shift pneumonia

Type of vaccines seasonal

vaccine, pandemic vaccine pre-pandemicvaccine

Seasonal vaccine : strain H1N1, H3N2 B strains (Northern hemisphere) (Southern hemisphere) antigenic drift

Pandemic vaccine : pandemic 4-6 4-6 industrial scale

Pre-pandemic vaccine : clinical trial SanofiPasteur 90 g hemagglutininantigen/1 dose GSK 3.75/1 dose Sanofi Pasteur adjuvant

Adjuvant aluminium hydroxide adjuvant GSK adjuvant AS03 SanofiPasteur MF59

151 :

H5N1 pandemic H5N1 fertilized egg pathogen free cell culture

pandemic pre-pandemic vaccine pre-pandemic vaccine H5N1 H1N1 2009 H5N1

2009H1N1

2009 H1N1 17 2552 , (.. 2552) 4,000

40 pandemic

Phase 1 : Phase 2 : Phase 3 : , Phase 4 : (small cluster) (WHO region) 2 (..2552 )Phase 5 :

152 :

(large cluster)Phase 6 : pandemic

H1N1

120 International centre International centre H1N1

wild type strain manufacturer H1N1 manufacturer process

Sanofi Pasteur H5N1 ( ) GSK 2 immunogenicity adjuvant ASO3 Sanofi Pasteur GSK pre-pandemic vaccine H1N1 H5N1 pre-pandemic vaccine

()

153 :

2 killed vaccine

life attenuated vaccineKilled vaccine Whole virus vaccine : pandemic

Split virus vaccine : Subunit vaccine : haemagglutinin (HA) neuraminidase (NA) local

systemic reaction adverse reactions Guillain-Barre Syndrome complication

154 :

155 :

Allergen vaccine Allergen vaccine

(skin test) allergen extract 10 allergen vaccine allergen vaccine therapeutic vaccine Immunotherapy

allergen vaccine (allergicrhinitis), (allergicconjunctivitis), (atopic asthma) (stinging insect allergy lethal allergy ) stinging insect allergy allergy

2541 al lergenimmunotherapy allergen specificimmunotherapy

3.7 Allergen vaccine. .

3.

Allergen source

extraction

purification

standardization

Allergen extract ~ Allergen vaccine

Skin test Immunotherapy

ENT Siriraj

156 :

allergen vaccine (allergen)

allergenvaccine 2454 2 Dr. LeonardNoon Dr. John Freeman desensitization (toxin) (neutralization) hyposensitization 2 immunotherapy

allergen vaccine 2454 immunology

(pollen) pollen 40 placebo control trial

(immunology)

2550 1, 7, 1, 6 1-3

- mast cell basophil degranulation anaphylaxis systemic reaction 1 mast cell basophil

- T cell tolerance Tregulatory cell Th2 activity T cell tolerance cell reactivity T celltolerance

- mast cell eosinophil mediator late-phase response

skin test reactivity

- skin test reactivity

157 :

Immunologic changes during the course of allergen-SIT

- IgE 1 skin testreactivity IgE 1 IgE skin test reactivity

- blocking antibody specificIgG4 6 IgA IgG1

antigen presenting cell T cell (To) Th2

cytokine interleukin 4 (IL-4) B cell IgE allergen allergicresponse 2

1) immune deviation Th2 Th1 Th1 interferon (IFN-) B cell blocking antibody (IgG)

2) T cell regulatory T cell (Tr1) allergic response

: Akdis M, Akdis C. JACI 2007; 119: 780-9

158 :

cytokine 2 IL-10 blocking antibody (IgG) TGF- eosinophil Th2 activity

: Akdis C, Akdis M. JACI 2009; 123: 735-46.

allergic response

Regulatory T cell regulatory T cell Th17 bacterial infection

regulatory T cell

159 :

allergenvaccine

1. T cell response immune deviation Th2 allergy Th1

2. effector cell basophil, mast cell eosinophil tolerance

3. antibody responses specific IgE blockingantibody IgG1, IgG4 IgA

allergen vaccine allergen vaccine - (raw material)

source material allergen vaccine source material (foreign substances)

- source material-

(process) (storage)Allergen vaccine

50-100

(House dust mite): - Pure mite body (PMB)

- Whole mite culture (WMC)

(culture medium)

pure mite body whole mite culture

(Insects) :

-