:
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) :
-