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Functional antibody activity as measured by opsonophagocytosis. Sandra Romero-Steiner, Ph.D. Respiratory Diseases Immunology Laboratory. Induction of antibodies. Disease Vaccination Passive immunization Colonization Cross reactive. Antibody Measurements. Quantitative measurement - PowerPoint PPT Presentation
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Functional antibody activity Functional antibody activity as measured by as measured by
opsonophagocytosisopsonophagocytosis
Sandra Romero-Steiner, Ph.D.Sandra Romero-Steiner, Ph.D.Respiratory Diseases Respiratory Diseases
Immunology LaboratoryImmunology Laboratory
Induction of antibodiesInduction of antibodies
DiseaseDisease VaccinationVaccination Passive Passive
immunizationimmunization ColonizationColonization Cross reactiveCross reactive
Antibody MeasurementsAntibody Measurements Quantitative measurement Quantitative measurement
(ELISA) = (ELISA) = g/mlg/ml Functional determinationsFunctional determinations
How well do those Abs protect?How well do those Abs protect? Animal protection Animal protection OpsonophagocytosisOpsonophagocytosis
Indicator of MemoryIndicator of Memory Antibody avidityAntibody avidity B-cell stimulation (Elispot)B-cell stimulation (Elispot)
OpsonophagocytosisOpsonophagocytosis
OPA PlayersOPA Players
FcR CR1/CR3
+
PMN
C3b/iC3b
Ab
Bacteria
OpsonopaghocytosisOpsonopaghocytosis
Romero-Steiner Romero-Steiner et alet al. . CDLI 1997;4:415-22CDLI 1997;4:415-22 Four componentsFour components
SerumSerum BacteriaBacteria ComplementComplement Culturable phagocytesCulturable phagocytes
Internalized Pnc are killed Internalized Pnc are killed OPA titer OPA titer
VIABILITY OPA
Unk1 Unk2 Unk3 Unk4 QC Unk1 Unk2 Unk3 Unk4 QC globulinglobulin
C’ controlsC’ controls
1:81:81:161:161:321:32 .. .. .. ..1:10241:1024
1:641:64 . . .. .. .. ..20482048
OPA titer determination OPA titer determination (50% (50% killing)killing)
Tom Taylor, PAOAW, June 2005Tom Taylor, PAOAW, June 2005
1/Serum Dilution
Midpoint(50% Killing)
Continuous Titer 50%
0%
100%
Measu
red
Killin
g
DiscontinuousTiter >50%
8 16 32 64 128 256
Killing OPAsKilling OPAs
Single serotypeSingle serotype Time consuming Time consuming Reagent demandingReagent demanding
Multivalent assaysMultivalent assays 2 serotypes2 serotypes 4 serotypes4 serotypes 7 serotypes7 serotypes
SOPA vs MOPA-4 SOPA vs MOPA-4 (Burton, R. PAOAW, June (Burton, R. PAOAW, June
2005)2005) Pn 9V
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000SOPA
MO
PA-4
R2=0.95 (R2=0.94)
Pn 18C
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000SOPA
MO
PA-4
R2=0.98
Pn 19F
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000SOPA
MO
PA-4
Pn 23F
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000SOPA
MO
PA-4
R2=0.98 R2=0.60
(R2=0.99)
Automation of killing Automation of killing OPAsOPAs
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7 8sample dilutions
CF
U/s
po
t
P49C P50C P51C P52C
P53C P54C TP Max
vOPA fOPA
Bieging, K. et al.Bieging, K. et al. CDLI 2005; 10:1238-42CDLI 2005; 10:1238-42
r = 0.76 to 0.97 r = 0.76 to 0.97 7-valent fOPA7-valent fOPA
Bogaert, D. et al, Bogaert, D. et al, Vaccine 2004; 22:4014-20Vaccine 2004; 22:4014-20
Uptake OPAsUptake OPAs
Flow cytometric methodsFlow cytometric methods Uptake vs killingUptake vs killing
Killed bacteriaKilled bacteria Ps-coated particlesPs-coated particles
Single serotypeSingle serotype Multivalent assays Multivalent assays
4 serotypes4 serotypes
Flow cytometric OPA Flow cytometric OPA (Uptake of killed bacteria or Ps-coated (Uptake of killed bacteria or Ps-coated
particles)particles)Data.011
0 200 400 600 800 1000SSC-Height
R1
Data.011
100 101 102 103 104
FL2-Height
M1
Data.107
100 101 102 103 104
FL2-Height
M1
Phagocytic cellsPhagocytic cells Complement controlComplement control
Positive RxPositive Rx
Example OPA Graph
Reciprocal Dilution
8 16 32 64 128 256 5121024
Per
cen
t U
pta
ke
0
20
40
60
80
100
Pt Sample
Complement uptake
50% Maximum Uptake
Reported Titer
OPA titerOPA titerMartinez, J. et al. CDLI 1999; 6:581-6.
uptake
Multiplexed Flow OPAMultiplexed Flow OPAExample OPA Graph
Reciprocal Dilution
8 16 32 64 128 256 5121024
Perc
ent U
ptak
e
0
20
40
60
80
100
Pt Sample
Complement uptake
50% Maximum Uptake
Reported Titer
MonovalentMonovalent
uptake
uptake
Uptake
uptake
Multiplexed Bead Based OPA
Log2 Reciprocol Dilution
2 4 6 8 10P
erce
nt U
ptak
e by
HL6
0 P
MN
s5
10
15
20
25
30
35
40
45
Serotype 14, GreenSerotype 9V, PinkSerotype 6B, RedSerotype 4, Far Red
MultivalentMultivalent
Martinez, J. et al. submitted to CDLI, 2005 and funded by Flow Applications
r = 0.53 to 0.95r = 0.53 to 0.95
r = 0.61 to 0.91r = 0.61 to 0.91
r = 0.68 to 0.88r = 0.68 to 0.88
Current validation statusCurrent validation status
Single serotype killing assaySingle serotype killing assay Developed, standardized, evaluated Developed, standardized, evaluated
and validated at the GLP leveland validated at the GLP level Multivalent killing assaysMultivalent killing assays
Developed and standardizedDeveloped and standardized Fluorescent killing assaysFluorescent killing assays
Developed and standardizedDeveloped and standardized Flow cytometric (uptake) assaysFlow cytometric (uptake) assays
Developed and standardizedDeveloped and standardized
Multi-laboratory EvaluationMulti-laboratory Evaluation Romero-Steiner Romero-Steiner et al.et al. CDLI 2003; 10:1019-24 CDLI 2003; 10:1019-24
S. S. pneumoniapneumonia
eeSerotypeSerotype
Lab 1 Lab 2 Lab 3 Lab 4 Lab Lab 1 Lab 2 Lab 3 Lab 4 Lab 55
4 88a, 100b 79, 88 100, 100 96, 96 96, 100
6B 75, 88 83, 100 56, 72 83, 88 88, 92
9V 63, 71 83, 88 75, 94 83, 96 83, 92
14 63, 83 75, 83 81, 94 92, 92 88, 92
18C 88, 96 88, 96 56, 69 88, 92 88, 100
19F 79, 96 88, 96 94, 100 96, 100 88, 100
23F 100, 100 100, 100
100, 100 96, 100 92, 96
a Within one dilution about the median OPA titer – 75% overall b Within two dilutions about the median OPA titer – 88% overall
Romero-Steiner Romero-Steiner et alet al. CDLI 2003;10:1019-24. CDLI 2003;10:1019-24
Higher agreement in sera with Higher agreement in sera with low titerslow titers Lower agreement in sera with high titersLower agreement in sera with high titers
Multi-laboratory EvaluationMulti-laboratory Evaluation
3803
2938
1039
3803
1838
0773
3803
4238
1399
3802
9238
0807
3803
7638
0847
3803
7238
0964
3803
6038
0828
3803
3038
0638
3803
8638
0859
3803
2738
0808
3803
5138
0824
3802
9838
0860
Log
2 O
PA
Tit
er
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Type 14 D
Validation of killing OPAValidation of killing OPA B. T. Hu et al. CDLI 2005, 12: 287-295.B. T. Hu et al. CDLI 2005, 12: 287-295.
Specificity Specificity >> 80% 80% ((>> 87% CDLI, 1997) 87% CDLI, 1997) Heterologous Ps Heterologous Ps << 20% 20%
Intermediate PrecisionIntermediate Precision Overall 81% of titers within 2 dil. of medianOverall 81% of titers within 2 dil. of median
Linearity (9 serotypes)Linearity (9 serotypes) r = 0.982 to 1.000, slopes = -0.850 to -1.350r = 0.982 to 1.000, slopes = -0.850 to -1.350
Accuracy (9 serotypes)Accuracy (9 serotypes) All types 100% except 14 (81%) & 23F (75%)All types 100% except 14 (81%) & 23F (75%)
RobustnessRobustness
OPA as a correlate of OPA as a correlate of protectionprotection
ELISA in healthy populationsELISA in healthy populations Passive protection in animalsPassive protection in animals Minimum level needed for Minimum level needed for
vaccine efficacy in infantsvaccine efficacy in infants
Log2 ELISA concentration (g/ml)
-2 0 2 4 6 8 10
Log
2 O
PA
tit
er
0
2
4
6
8
10
12
14
Type 4Type 6BType 9VType 14Type 18CType 19FType 23FLinear regression
r = 0.79slope = 1.2n = 49
Figure 3.
Infant mice nonbacteremic at 48 hours (%)
0 20 40 60 80 100
Lo
g 2 p
roje
cte
d O
PA
tit
er
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
n = 731r = .84P < .001
Johnson, et al. JID 1999; 180:133Johnson, et al. JID 1999; 180:133
Protective OPA titer in aProtective OPA titer in amouse passive protection mouse passive protection
modelmodel Type 1 Type 4 Type 5 Type 6B Type 18C Type 23
OPA = 8OPA = 8
75%75%
0
20
40
60
80
100
0.01 0.1 1 10 100
Ab Concentration
% >
Ab
Co
nce
ntr
atti
on
Vax
Control
97.9
12.9
VE = 1 - (1-.979) (1-.129)
= .976
[Ab] for 97.9% protection in the target population = 0.20 [Ab] for 97.9% protection in the target population = 0.20 µg/mlµg/ml
~0.2g/ml
Reverse cumulative distributions of post dose 3Reverse cumulative distributions of post dose 3 ELISA Ab for 7 serotypes in infantsELISA Ab for 7 serotypes in infants
(Black, et al., North Calif. Trial)
Data from Dr. Kohberger, WHO 2003
0.01 0.1 1 10
ELISA Concentration (g/ml)
1
10
100
1000
10000
OP
A T
ITE
R
7VPnC
Control
R = 0.92( p < 0.0001)
Total N = 79
ELISA concentration of 0.20 g/ml OPA titer of 1:8
Jodar, et al. Vaccine 2003; 21:3265-Jodar, et al. Vaccine 2003; 21:3265-32723272
Correlation of ELISA and OPA Correlation of ELISA and OPA (North CA KP infant study)(North CA KP infant study)
1:8
0.2 g/ml
Functional Abs in the elderlyFunctional Abs in the elderly
Protective levels are unknownProtective levels are unknown Reduced function in elderly receiving PPV-Reduced function in elderly receiving PPV-
23 (CID 1999;29:281-8)23 (CID 1999;29:281-8) OPA (80-89 years & OPA (80-89 years & >>90 years)90 years) Lower Ab avidityLower Ab avidity Poor correlation with ELISA IgG (r=0.3 to Poor correlation with ELISA IgG (r=0.3 to
0.8)0.8) Do not protect miceDo not protect mice
Usinger and Lucas I&I, 1999;67:2366-70Usinger and Lucas I&I, 1999;67:2366-70 Absorption of cross-reactive antibodies Absorption of cross-reactive antibodies
(I&A 2005; 2:1-10) (I&A 2005; 2:1-10)
OPA in clinical vaccine trialsOPA in clinical vaccine trialsEkstrom, N. PAOAW, June 2005Ekstrom, N. PAOAW, June 2005
Killing OPA (Romero-Steiner et al.1997)Killing OPA (Romero-Steiner et al.1997)
Various phase 2 studies with different Pnc-conjugate vaccines in Various phase 2 studies with different Pnc-conjugate vaccines in Finnish infants (Anttila et al. 1999)Finnish infants (Anttila et al. 1999)
7-valent PncCRM and PncOMPC in Finnish infants in The Finnish 7-valent PncCRM and PncOMPC in Finnish infants in The Finnish Otitis Media Vaccine Trial (FinOM)Otitis Media Vaccine Trial (FinOM)
11-valent PncDT in Filipino infants (Puumalainen et al. 2003)11-valent PncDT in Filipino infants (Puumalainen et al. 2003) 23-valent PS in HIV+ Ugandan adults (French et al. 2004)23-valent PS in HIV+ Ugandan adults (French et al. 2004) 11-valent PncDT in Finnish and Israeli infants (Wuorimaa et al. 11-valent PncDT in Finnish and Israeli infants (Wuorimaa et al.
2005)2005) 11-valent Pn-PD in Finnish infants (Nurkka et al. 2005)11-valent Pn-PD in Finnish infants (Nurkka et al. 2005)
Flow cytometric OPA (Martinez et al. 1999)Flow cytometric OPA (Martinez et al. 1999) 11-valent PncDT in Filipino infants (Lucero et al. 2004) 9-valent PncCRM in South-African infants (Mahdi et al. 2005)
Advantages of using OPAAdvantages of using OPA Laboratory correlate of protectionLaboratory correlate of protection Reduce numbers of efficacy studiesReduce numbers of efficacy studies Reference method (killing sOPA)Reference method (killing sOPA) www.vaccine.uab.eduwww.vaccine.uab.edu StandardizedStandardized ValidatedValidated Culturable phagocytesCulturable phagocytes High through put High through put GLP conditions GLP conditions Data analysisData analysis
Disadvantages of OPADisadvantages of OPA
In vitro correlateIn vitro correlate Laboratory facilitiesLaboratory facilities Training of technical staffTraining of technical staff Multiplex assays need Multiplex assays need
validationvalidation
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