Opsonization Assay:
Functional Correlate of Protection
Dr. George M. Carlone
CDC, Atlanta
Elie Metchnikoff
OVERVIEW
• Host protection
• Serologic markers measured in immunogenicity trials
• Opsonization – how does it work?
• Opsonic assay formats
• Why it’s important to measure functional activity
• Correlates we observe in animals & humans
Host protection against pneumococcal disease
is mainly mediated by phagocytosis
• Phagocytosis – general process describing the engulfment & destruction of extracellularly-derived materials by
phagocytic cells, such as macrophages & neutrophils
• Opsophagocytosis – opsonophagocytosis is a specific
mechanism by which the host protects against infection,
with the participation of serum opsonins
• Opsonins – antibody and complement (e.g., IgG and C3b)
• Functional antibody – leads to effective opsonization
and recovery from infection
Serologic markers that correlate with protection
in infants vaccinated with conjugate vaccine
•
•
•
•
IgG ELISA - WHO standardized and validated assay
protocol (www.vaccine.UAB.edu)
Opsonophagocytic assay (OPA) - CDC standard manual
killing assay (reference method) protocol
(www.vaccine.UAB.edu)
Antibody avidity – indicator of memory
maturation of antibody function & quality
Immunological memory - immune recall
(IgG, rapid, memory T & B cells)
** Evaluated in efficacy study
Comparison of serologic markers that correlate with
protection in infants and/or adults
Laboratory Assays Conjugate Vaccine Poly. Vaccine
Infant
Adult
Infant Adult
IgG ELISA
X
X
na
X
OPA
X
X
na
X
Avidity
X
?
na
NO
Memory
X
X?
na
NO
Animal Model
passive protect.
Cellular Immunity
cytokines, etc.
X
X
na
X
X
?
na
X
Opsonization - how does it work?
antigen
IgG
first complement
proteins
complement
cascade
cell swells
and bursts
complement inserts
into cell wall
IgG and C3b are known as opsonins and the
process of attachment is called opsonization
*subclasses differ in
C’ deposition capacity
Receptor
for IgG
Receptor for iC3b
(high & low avidity)
Phagocytic cell
www.cat.cc.md.us/.../ opsonization/u1fig26n.html
Opsonophagocytosis of pneumococci requires
bacteria, Ab, C’, and phagocytic cells
With Ab and C’
Without Ab and C’
Pnc are not being
engulfed
Pnc are being
engulfed & killed
www.lsumc.edu/campus/micr/opson.htm
Engulfment and killing
1. attachment
of bacteria / beads
phagocyte
2. engulfment
phagosome
4. respiratory burst
stimulation of NADPH
oxidase
phagolysosome
residual body
lysosomes
3. degranulation:
fusion of granules
to phagosome
www.cvm.uiuc.edu/.../ vp331/Intracell_Bacteria
bacterial killing
and digestion
Capsules are anti-phagocytic
(interferes with attraction of phagocyte,
engulfment and recognition of cell as foreign)
*The thicker the capsule the
more Ab required for OPA
www.uic.edu/.../ slide0231.htm
Opsonic Assay Formats
• Manual assays (viable cells) – measures killing
CDC reference method (Romero-Steiner),
multiplexed, antibiotic resistant (Kim, Nahm; Bogaert)
radiometric (Jonsdottir), other
• Flow cytometric (non-viable cells) – measures uptake
single color (Martinez; Jansen)
three color multiplex (Martinez)
bead based target multiplexed (Martinez), other
Why is functional (OPA) activity important
to measure for vaccine evaluation?
• Opsonophagocytic
activity correlates with protection,
however, Ab conc. may not always corr with function
• Formal efficacy trials will likely not be done
• Serotypes
in new vaccine formulations will not have
proven efficacy
• Vaccines
from different manufacturers will likely be
different (structurally, immunologically, etc.)
• Therefore,
functional activity is an essential
measurement for vaccine comparison
What do animal models tell us
about potential
correlates of protection?
Protective activity of serotype 6B specific IgG against
bacteremia after challenge with a 6B isolate
Absence of bacteremia 48hr after challenge
IgG 48hr after
immunization
Serum 89SF
#/total/(%)
Clinical sample
#/total/(%)
P value
0.0 ug/ml
2/37 (5.4)
5/55 (9.1)
0.7
0.05
6/16 (38)
19/35 (54)
0.4
0.1
23/29 (79)
24/24 (100)
0.03
0.2
17/17 (100)
9/9 (100)
NA
Johnson, et al. 1999. JId. 180:133.
Correlation between bacteremia and OPA titer in a
mouse passive protection model
`
6
Serotype 1
Serotype 4
Serotype 5
Serotype 6B
Serotype 18C
Serotype 23
5
Log
Log
OPA titer
2 OPA Titer
2 projected
4
1:8
3
2
1
0
-1
n = 731
r = .84
P < .001
-2
-3
-4
75%
-5
-6
0
20
40
60
80
Infant mice nonbacteremic
100
Infant miceat
nonbacteremic
48 hours (%) at 48 hr (%)
Johnson, et al. 1999. JId. 180:133.
What do human trials tell us
about potential
correlates of protection?
11-Valent Conjugate Vaccine Response in Filipino Infants
Serotype 6B
18 wk old
10 mo old
+ EIA & OPA
pre (6 wk)
OPA
OPA
Serotype 4
EIA
Serotype 19F
Regression…. 18 wk old
___10 mo old
OPA
OPA
EIA
Serotype 14
EIA
Puumalainen, et al. 2003. JID. 187:1704.
EIA
Reverse cumulative distributions of post dose 3
ELISA Ab for 7 serotypes in infants
(Black, et al., North Calif. Trial)
97.9
% >= Ab Conc
100
VE = 1 - (1-.979)
(1-.129)
= .976
80
60
Vax
Control
40
20
12.9
0
0.01
0.1
1
~0.2ug/ml
0.18
[Ab] prot
Data from Dr. Kohberger, WHO 2003
10
100
Ab Concentration
Ignoring Ab levels in controls obtains [Ab] prot = .20 µg/ml
Correlation of ELISA and OPA (North CA KP infant study)
Jodar, et al. 2003. Vaccine
Total N = 79
R = 0.80
(
p
0.0001)
R = <0.92
10000
( p < 0.0001)
7VPnC
Control
1000
R = 0.80
( p < 0.0001)
100
1:8
10
0.2ug/ml
1
0.01
0.1
1
10
ELISA Concentration (g/ml)
ELISA concentration of 0.20  OPA titer of 1:8
SUMMARY
• OPA is a correlate of protection
• Animal & human OPA protection data appear to agree
• ELISA & OPA are primary end-points (good correlation)
• OPA formats include killing (std.) and uptake assays
• Functional activity is influenced by avidity and age
• Immunogenicity may be influenced by study design
and/or vaccine formulation, dose, scheduling, etc.
• Formal efficacy trials will likely not be done