Opsonization Assay:

1
Opsonization Assay Functional Correlate of
Protection
Dr. George M. Carlone CDC, Atlanta
Elie Metchnikoff
2
OVERVIEW

• Host protection
• Serologic markers measured in immunogenicity
  trials
• Opsonization how does it work?
• Opsonic assay formats
• Why its important to measure functional
  activity
• Correlates we observe in animals humans

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Host protection against pneumococcal disease
is mainly mediated by phagocytosis

• Phagocytosis general process describing the
  engulf- ment 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

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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

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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. X X na X
Cellular Immunity cytokines, etc. X ? na X
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Opsonization - how does it work?
antigen IgG
first complement proteins
complement cascade
complement inserts into cell wall
cell swells and bursts
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IgG and C3b are known as opsonins and the process
of attachment is called opsonization
subclasses differ in C deposition capacity
Receptor for iC3b (high low avidity)
Receptor for IgG
Phagocytic cell
www.cat.cc.md.us/.../ opsonization/u1fig26n.html
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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
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Engulfment and killing
1. attachment of bacteria / beads
phagocyte
2. engulfment
4. respiratory burst stimulation of NADPH oxidase
phagosome
phagolysosome
residual body
lysosomes
3. degranulation fusion of granules to phagosome
bacterial killing and digestion
www.cvm.uiuc.edu/.../ vp331/Intracell_Bacteria
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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
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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

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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

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What do animal models tell us about
potential correlates of protection?
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Protective activity of serotype 6B specific IgG
against bacteremia after challenge with a 6B
isolate
Absence of bacteremia 48hr after challenge Absence of bacteremia 48hr after challenge 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. 180133.
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Correlation between bacteremia and OPA titer in
a mouse passive protection model
Serotype 1 Serotype 4 Serotype 5
Serotype 6B Serotype 18C Serotype 23
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Log2 OPA Titer
75
Infant mice nonbacteremic at 48 hr ()
Johnson, et al. 1999. JId. 180133.
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What do human trials tell us about
potential correlates of protection?
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11-Valent Conjugate Vaccine Response in Filipino
Infants
Serotype 4
Serotype 6B


18 wk old 10 mo old EIA OPA pre (6
wk)
OPA
OPA


EIA Serotype 14
EIA Serotype 19F



Regression- . 18 wk old ___10 mo old
OPA
OPA


EIA
EIA
Puumalainen, et al. 2003. JID. 1871704.
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Reverse cumulative distributions of post dose 3
ELISA Ab for 7 serotypes in infants (Black, et
al., North Calif. Trial)
97.9
VE 1 - (1-.979) (1-.129)
.976
12.9
0.18 Ab prot
0.2ug/ml

Data from Dr. Kohberger, WHO 2003
Ignoring Ab levels in controls obtains Ab prot
.20 µg/ml
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Correlation of ELISA and OPA (North CA KP infant
study)
Total N 79
Jodar, et al. 2003. Vaccine
R 0.80 ( p lt 0.0001)
R 0.92 ( p lt 0.0001)
10000
7VPnC
Control
R 0.80 ( p lt 0.0001)
1000
OPA TITER
100
18
10
0.2ug/ml
1
0.01
0.1
1
10
ELISA Concentration (?g/ml)
ELISA concentration of 0.20 ? OPA titer of 18
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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