opa methods in clinical vaccine trials: experience with killing and flow cytometric opa methods nina...
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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