OPA methods in clinical vaccine trials: experience with killing and flow

cytometric OPA methods

Nina EkstromVaccine Immunology Laboratory

National Public Health Institute (KTL)Helsinki, Finland

OPA in clinical vaccine trials

• Killing type OPA (Romero-Steiner et al.1997)

– Various phase 2 studies with different Pnc-conjugate vaccines in Finnish infants (Anttila et al. 1999)

– 7-valent PncCRM and PncOMPC in Finnish infants in The Finnish Otitis Media Vaccine Trial (FinOM)

– 11-valent PncDT in Filipino infants (Puumalainen et al. 2003)– 23-valent PS in HIV+ Ugandan adults (French et al. 2004)– 11-valent PncDT in Finnish and Israeli infants (Wuorimaa et al. 2005)– 11-valent Pn-PD in Finnish infants (Nurkka et al. 2005)

• 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)

The Finnish Otitis Media Vaccine Trial(FinOM)

• Randomized, double-blind, phase 3 cohort study designed to evaluate in parallel two 7-valent Pnc conjugate vaccines (N= 2497 Finnish infants)

• Study vaccines ( 2, 4, 6 and 12 months of age)

– PncCRM (Wyeth Vaccines) N=835– PncOMPC (Merck & Co. Inc.) N= 831– Hepatitis B (Merck & Co. Inc.) N=831

• Efficacy of two PCVs against serotype-specific pneumococcal acute otitis media (AOM) compared with control vaccine

• Immunogenicity• Quality of antibodies (avidity)• Functionality of antibodies (OPA)

choice of OPA method• Serological correlates of protection

Aims of the FinOM Vaccine Trial

Comparison of OPA methods

• Killing type OPA (Romero-Steiner et al.1997)• Radio-OPA (Vidarsson et al.1994)• Flow cytometric OPA-1 (Jansen et al.1998)• Flow cytometric OPA-2 (Martinez et al.1999)

• Sera from infants (n=10-16) immunized at 2, 4, 6 with heptavalent PncCRM and at 15 mo with PncCRM or 23-valent PncPS

• Pnc serotypes 6B and 19F (reference strains from CDC)• IgG concentrations were determined by EIA

Differences in OPA protocols

Killing assay Radio assay Flow assay 1 Flow assay 2

Bacteria Live, untreated, grown once to log phase (1 x log)

Live, radiolabelled (H3), grown 1 x log

Killed, labelled with FITC, grown 3 x log

Killed, labelled with 5,6-carboxyfluorescein succinimidyl ester, grown 1 x log

Phagocytes Fresh PMNLs Fresh PMNLs Fresh PMNLs HL-60 cells

Bact : Phag ratio 1:400 10:1 10:1 4:1

Complement source

Baby rabbit serum Pooled serum from hypo- and agamma- globulinemic patients (6B), IgG-depleted serum from a healthy adult (19F)

IgG-depleted human pooled serum

Baby rabbit serum

Complement concentration (%)

12.5 5 (6B), 12 (19F) 2 12.5

Vakevainen et al. CDLI 2001

Relationship between killing, radio and flow-1 OPAs

Killing vs . Radio6B

1

10

100

1000

1 10 100 1000 10000 100000

killing OPA

rad

io O

PA

m7; r=0.92, p< .001m15; r=0.78, p< .001

m16; r=0.76, p< .01

r=0.95p< .001

Killing vs . Flow 6B

1

10

100

1000

1 10 100 1000 10000 100000

killing OPA

flo

w-1

OP

A

m7; r=0.39, p=0.13m15; r=-0.14, p=0.62

m16; r=0.82, p< .001

r=0.78p< .001

Radio vs . Flow-16B

1

10

100

1000

1 10 100 1000

radio OPA

flo

w-1

OP

A

m7; r=0.43, p=0.09m15; r=-0.08, p=0.77

m16; r=0.82, p< .001

r=0.82p< .001

19F

1

10

100

1000

1 10 100 1000 10000

killing OPA

rad

io O

PA

m7; r=0.81, p< .001

m15; r=0.83, p< .001

m16; r=0.78, p< .001

r=0.84

p< .001

19F

1

10

100

1000

1 10 100 1000 10000

killing OPA

flo

w O

PA

m7; r=0.27, p=0.31

m15; r=0.05, p=0.85

m16; r=0.54, p< .05

r=0.50p< .001

19F

1

10

100

1000

1 10 100 1000

radio OPA

flo

w O

PA

m7; r=0.35, p=0.19

m15; r=-0.04, p=0.89

m16; r=0.37, p=0.15

r=0.55p< .001

6B

19F

Vakevainen et al. CDLI 2001

Relationship between OPA and EIA

Radio vs . EIA 6B

OP

A (

rad

io)

m7; r=0.84, p< .001m15; r=0.72, p< .01m16; r=0.75, p< .01

Killing vs . EIA6B

1

10

100

1000

10000

100000

OP

A (

kill

ing

)

m7; r=0.91, p< .001m15; r=0.70, p< .01m16; r=0.81, p< .001

Flow 1 vs . EIA 6B

OP

A (

flo

w 1

)

m7; r=0.51, p< .05m15; r=-0.34, p=0.19

m16; r=0.84, p< .001

19F

1

10

100

1000

10000

100000

0,1 1 10 100 1000IgG (g/ml)

OP

A (

kill

ing

)

m7; r=0.79, p< .001m15; r=0.89, p< .001m16; r=0.91, p< .001

19F

0,1 1 10 100 1000IgG (g/ml)

OP

A (

rad

io)

m7; r=0.72, p< .01m15; r=0.67, p< .01m16; r=0.74, p< .01

19F

0,1 1 10 100 1000IgG (g/ml)

OP

A (

flo

w 1

)

m7; r=0.39, p=0.13m15; r=0.06, p=0.83m16; r=0.53, p< .05

6B

19F

Vakevainen et al. CDLI 2001

Flow-2 OPA vs. other OPA methods

Flow 2 vs . Killing6B

y = 1,5869x0,9543

r=0.97, p< .001

1

10

100

1000

10000

100000

OP

A (

kill

ing

)

Flow 2 vs . Radio6B

y = 0,3708x0,7912

r=0.94, p< .001

OP

A (

rad

io)

Flow 2 vs . Flow 16B

y = 0,4096x0,6543

r=0.89, p< .001

OP

A (

flo

w 1

)

19F

y = 1,6177x1,061

r=0.92, p< .001

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000OPA (flow 2)

OP

A (

kill

ing

)

19F

y = 0,7296x0,698

r=0.77, p< .001

1 10 100 1000 10000 100000

OPA (flow 2)

OP

A (

rad

io)

19F

y = 1,0654x0,3886

r=0.47, p< .05

1 10 100 1000 10000 100000OPA (flow 2)

OP

A (

flo

w 1

)

6B

19F

Vakevainen et al. CDLI 2001

Summary of comparisons

• Different OPAs gave comparable results– levels of OPA were different– serotype-specific differences (19F > 6B)

• OPA correlated with IgG concentration– Killing, radio > flow-1

• Highest correlation between killing and radio OPAs• Differences in sensitivity (emphasized for 19F)

– Killing > Radio > Flow-2 > Flow-1

• The Killing OPA was chosen to be used in the FinOM Trial

+ Most sensitive+ Measured the killing of bacteria+ Standardised, ”the golden standard”+ Reproducibility between laboratories had been evaluated in the

multilaboratory study (Romero-Steiner et al. 2003)- Laborious- Slow (max. 90 analyses/week)- Long-term repeteability ? ± Price

The FinOM Trial - OPA analyses

• N = 166 infants in Kangasala cohort

– PncCRM N = 56– PncOMPC N = 52– Control (Hepatitis B) N = 58

• Immunizations at 2, 4, 6 and 12 months of age

• OPA was performed for 7, 12, 13 and 24 mo samples

• OPA for serotypes 6B, 19F and 23F

• Killing-OPA as described by Romero-Steiner et al. 1997

– Differentiated HL-60 cells as effector cells– S. Pneumoniae reference strains from CDC– Baby rabbit complement– Colonies counted manually

• IgG concentrations were determined by EIA

The FinOM Trial - Killing OPA

IgG concetrations & OPA titers in The FinOM Trial

6B

0,01

0,1

1

10

100

age (mo)

GM

C (

ug

/ml)

7 12 13 24

19F

0,01

0,1

1

10

100

age (mo)

GM

C (

ug

/ml)

7 12 13 24

23F

0,01

0,1

1

10

100

age (mo)

GM

C (

ug

/ml)

PncCRM

PncOMPC

Control

7 12 13 24

6B

1

10

100

1000

age (mo)

GM

OP

A

7 12 13 24

19F

1

10

100

1000

age (mo)

GM

OP

A

7 12 13 24

23F

1

10

100

1000

age (mo)

GM

OP

A

PncCRM

PncOMPC

Control

7 12 13 24

IgG concentration (ng/ml) required to kill 50% of Pnc (EIA:OPA ratio)

Serotype-specific efficacy % against AOM

ng/ml neened for 50% killing

Age (mo) Vaccine 7 13 24

6B PncCRM 84 10 13 17

PncOMPC 79 9* 13 25*19F PncCRM 25 97 59 81

PncOMPC 37 50 78 104

23F PncCRM 59 59 31 58PncOMPC 52 92* 96 65*

* < 50% infants had a detectable OPA titer

Correlation between EIA & OPA

7 mo

13 mo

6B 19F 23F

19F 7 months

1

10

100

1000

10000

0 0 1 10 100EIA

OP

A

PncCRM r= 0.76

PncOMPC r= 0.61

23F 7 months

1

10

100

1000

10000

0,01 0,1 1 10 100EIA

OP

A

PncCRM r= 0.72

PncOMPC r= -0.04

23F 13 months

1

10

100

1000

10000

0,01 0,1 1 10 100

EIA

OP

A

PncCRM =0.88

PncOMPC r= 0.84

19F 13 months

1

10

100

1000

10000

0 0 1 10 100 1000EIA

OP

A

PncCRM r= 0.57

PncOMPC r= 0.86

6B 7 months

1

10

100

1000

10000

0 0 1 10 100EIA

OP

A

PncCRM r=0.5

PncOMPC r= 0.32

6B 13 months

1

10

100

1000

10000

0 0 1 10 100

EIA

OP

A

PncCRM r= 0.6

PncOMPC r= 0.29

OPA as a correlate of protection

• Our results conform that Ab concentration is the primary correlate of protection, but that OPA is needed as a secondary correlate of protection:

– Despite equal Ab concentrations the functionality of Ab’s may differ between serotypes

– EIA:OPA ratio seems to correlate better with protection against AOM than Ab concentration at population level

Experience with the flow cytometric OPA method

+ Correlates with killing OPA+ Rapid and less laborious+ Unaffected by antibiotics in

sera of e.g HIV+ persons+ Multiplexing possible- Long-term repeteability ?- Flow cytometer needed± Price 1

10

100

1000

10000

1 10 100 1000 10000

killing assay

MP

-OP

A

19F N=41r=0.64

23F N=39r=0.75

19F: Alexa Fluor Dye 64723F: 5,6-carboxyfluorescein, succinimidyl ester

1

10

100

1000

10000

1 10 100 1000 10000Killing OPA

FA

CS

-OP

A

6B r = 0.86 19F r = 0.88 23F r = 0.88 4 r = 0.86

All r = 0.80

Quantitative and Qualitative Antibody Response to Pneumococcal Conjugate Vaccine Among African Human Immunodeficiency Virus-Infected and Uninfected Children

Madhi SA †, Kuwanda L*, Cutland C †, Holm A ‡, Kayhty H ‡, and Klugman KP §

*National Institute of Communicable Diseases/University of the Witwatersrand/Medical Research Council: Respiratory and Meningeal Pathogens Reasearch Unit, and the †Paediatric Infectious Diseases Research Unit, Wits Health Consortium, University of the Witwatersrand, Johannesburg, South Africa; the ‡National Public Health Institute, Helsinki, Finland; and the Departments of § International Health, Rollins School of Public Health and Infectious Diseases, School of Medicine, Emory University, Atlanta, GA

PIDJ 2005;24:410-16

Experience with the flow cytometric OPA method – Soweto Trial

Soweto Trial

• Nested study of a larger phase 3 trial that evaluated the efficacy of a 9-valent PncCRM in 39 836 children

• Study vaccine or placebo were given at 6, 10 and 14 weeks of age• Blood sample was taken a month after the 3rd vaccine dose• EIA and OPA analyses were performed at KTL, Finland• OPA analyses were performed using the flow cytometric OPA assay

(Martinez et al. 1999)– HL-60 cells as effector cells– S.pneumoniae strains from CDC– Baby rabbit complement

N = HIV + HIV -

PncCV 30 63

Placebo 36 64

Anti-Pnc in HIV + and HIV – children Madhi et al. PIDJ 2005

0,01

0,1

1

10

1 4 5 6B 9V 14 18C 19F 23F

HIV+PCV+HIV-PCV+HIV+PCV-HIV-PCV-

Serotype HIV+PCV+ HIV-PCV+ p

6B % ≥8 78 96 0.01

ng/ml needed for 50 % uptake

26 4 0.0005

19F % ≥8 46 91 0.00002

ng/ml needed for 50 % uptake

102 89 0.69

23F % ≥8 57 93 0.002

ng/ml needed for 50 % uptake

83 41 0.14

IgG concentration (ng/ml) required for 50% uptake Madhi et al. PIDJ 2005

Conclusion

• Despite equal Ab concentrations PCV induced Ab’s of HIV+ and HIV- children had different functional activity

• Abs produced by HIV+children were dysfunctional

• The results suggest that although Ab concentration is the primary correlate of protection OPA is needed as a secondary correlate of protection

Summary – OPA methods at KTL

Killing Flow OPA

Multiplex Flow OPA

Small sample volume + + +++High sensitivity +++ ++ ++

Easy to perform + ++ +++Repeteability/ Reproducibility ++ ++ ++Rapidity - ++ +++Antibiotics do not interfere - +++ +++Low cost + + +++

Acknowledgments

Vaccine Immunology Laboratory &Clinical Unit, Department of Vaccines, KTL:

Merja VakevainenHannele LehtonenMaijastiina KarpalaKaisa JousimiesMerja RyynanenNina NikkanenJukka JokinenHelena KäyhtyThe FinOM Study Group

CDC, Atlanta:

Sandra Romero-SteinerJoseph MartinezGeorge M. Carlone

Eijkman-Winkler Institute for Microbiology, Utrecht University Hospital, The Netherlands:

Wouter JansenAndre VerheulHarm Snippe

National University Hospital, Reykjavik,Iceland:

Eirikur SaelandIngileif Jonsdottir

Paediatric Infectious Diseases Research Unit, , University of theWitwatersrand, Johannesburg, South Africa

The group of Shabir Mahdi