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