Laboratory Tools for Evaluation of Pneumococcal Vaccine Immunogenicity

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Laboratory Tools for Evaluation of Pneumococcal
Vaccine Immunogenicity
George M. Carlone CDC, Atlanta, Georgia USA
ELISA/Avidity
Memory
Opsonic Activity
Animal Models
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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 CDC standard manual
  assay (reference method) Protocol
  www.vaccine.UAB.edu
• Immunological memory long-term protection
• Immune recall (IgG, rapid, memory T B cells)
• Antibody avidity good indicator of
  memory Maturation of antibody function quality
• Evaluated in efficacy study

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Comparison of Serologic Markers that Correlate
with Protection in Infants and/or Adults
Assays Conjugate Vaccine
Poly. Vaccine
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Quantitation of pneumococcal antibodies (ELISA -
not as simple as we thought)

• Farr assay (Schiffman 1980)
• - antibody binding to radiolabeled 14C-Ps
• - low to modest throughput large serum vol.
• - detects all classes and isotypes of antibody
• - serotype specificity in doubt
• ELISA (early 1980s)
• - poor corr. with efficacy animal protection
• - required 3 generations to get it optimized

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Evolution of pneumococcal ELISAs

• Ist generation
• - Ps antibody concentration overestimated
• - adsorption of serum with C-Ps (Musher, 1990)
• 2nd Generation
• - preadsorption of serum with C-Ps
• - still poor specificity in unimmunized adults
• (Coughlin and Carlone, 1998)
• 3rd Generation
• - Ps contamin. adsorbed with 22F Ps
• - highly serotype specific (WHO 2000 Frasch)

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Reverse Cumulative Distributions of Pooled Post
Dose 3 ELISA Antibody Conc. for 7 Serotypes
(Aggregate Data from 3 Studies) in Infants
Immunized unweighted pooling
Predicted VE
North CA KP Navajo South Africa
Unimmunized Unweighted pooling
0.35
0.35
Pooled VE 93.0
Predicted VE with 0.35 ?g/mLcutoff 92.5
Data from Dr. Kohberger, WHO 2003
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Future Direction Multiplex ELISA using Luminex
System (automated with high throughput)
Streptococcus pneumoniae polysaccharides covalentl
y bound to individual beads
Pickering, et al. 2002
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Opsonophagocytic Assay (functional antibody)

• Reference method, killing assay (Romero-Steiner,
  1997) - Multilab study submitted for
  publication - Protocol www.vaccine.UAB.edu
• Good to moderate correlation with ELISA IgG
• Target cells (HL-60 cells or donor cells)
• Antibiotic resistance, killing assay (Kim, et
  al. 2003)
• CDC Flow cytometric OPA, uptake assay
  (multiplexed)

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Why is functional (OPA) activity important to
measure during vaccine evaluation?

• Opsonic activity correlates with protection
• Formal efficacy trials will be limited or not
  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 evaluation

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Correlation of ELISA and OPA (North CA KP infant
study)
Jodar, et al. 2003. Vaccine
Total N 79
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
10
1
0.01
0.1
1
10
ELISA Concentration (?g/ml)
ELISA concentration of 0.20 ? OPA titer of 18
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Opsonic GMTs for young and old adults
vaccinated once with a polysaccharide vaccine
Romero-Steiner, 1999. CID, 29281
Young
63-79 Yr
Opsonophagocytic GMT
Serotype
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Opsonic GMTs ELISA GMCs for older children and
young adults with sickle cell disease - conjugate
vaccine boosted with Ps
Serotype 9V
Serotype 6B
Serotype 4
Opa
Opa
Opa
ELISA
ELISA
ELISA
...

Serotype 19F
Serotype 18C
Serotype 14
GMT (opsonic titer)
Opa
ELISA
GMC IgG ELISA (ug/ml)
Opa
Opa
ELISA
ELISA
...

Pre Post 7V 1 Post 7V 2 Post 23V
Pre Post 23V
Serotype 23F
Opa
ELISA
Vernacchio, et al. 2000. JID 181.

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Memory in Infants
GMC antibodies in infants who were immunized with
an 11-valent CV with or without alum at 2, 4, and
6 mo. of age and who received a booster at 12 mo.
(all booster responses, (P lt .05)
Wuorimaa, Vaccine, 200119
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Young adult antibody response after one dose of
conjugate and boosted with polysaccharide vaccine
Powers, 1996. JID 1731014
Wyeth-Lederle vaccine
Antibody Level (ug/ml)
CV
Ps
Months
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Young adult antibody response after two doses of
conjugate and boosted with polysaccharide vaccine
Kroon, 2001. Vaccine, 19886
Merck vaccine
Antibody Level (ug/ml)
CV
Ps
CV
Months
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Avidity

• Avidity maturation good indicator of memory in
  infants - Limited data on usefulness in adults
• Chaotropic agent added to ELISA format
• Inhibition of binding or elution of antibody
  formats
• Concentrations are in arbitrary units, not µg/ml

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Infant Avidity
Mean avidity indices 95 CIs in infants who
were immunized with an 11-valent CV
with/without alum at 2, 4, and 6 months of
age and who received a CV booster at 12 mo. of
age.
Wuorimaa, Vaccine, 200119
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Avidities After a Single Dose of Conjugate
Vaccine in Adults
Extracted from Wuorimaa, Vaccine, 200119
28 days
14 days
0 days
Avidity Index ()
Ps 1 Ps 5 Ps 6B Ps 14 Ps
19F Ps 23F
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Avidities in Adults After a Single Dose of
Conjugate and a Booster Dose of Polysaccharide
Vaccine
Wyeth-Lederle vaccine
Extracted from Wuorimaa, Vaccine, 200119
Avidity Index ()
DAYS Postvaccination
Alum gave slight increase
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SUMMARY

• Limited data on adults vaccinated with conjugate
  vaccine
• Not all markers used for infants are suitable
  for adults
• ELISA and OPA are primary end-points
• Functional activity is influenced by age
• Avidity maturation may not be a good marker in
  adults
• Immunogenicity may be influenced by study
  design and/or vaccine formulation dose,
  scheduling, etc.