# Presentation: 548990

## Laboratory Tools for Evaluation of  Pneumococcal Vaccine Immunogenicity

**Laboratory Tools for Evaluation of  Pneumococcal Vaccine Immunogenicity**

**George M. Carlone**
**CDC, Atlanta, Georgia USA**

**ELISA/Avidity**

**Opsonic Activity**

**Memory**

**Animal Models**

## Serologic Markers that Correlate with Protection in Infants Vaccinated with Conjugate Vaccine   

**IgG ELISA ****	****WHO standardized and validated assay****	****Protocol – **_[www.vaccine.UAB.edu](http://www.vaccine.uab.edu/)__  _**   **** **
**Opsonophagocytic assay ****    ****	****CDC standard manual assay (reference method)****	****Protocol – **_[www.vaccine.UAB.edu](http://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**

##         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 |

**Comparison of Serologic Markers that Correlate with  Protection in ****Infants**** and/or ****Adults**** **

## Quantitation of pneumococcal antibodies

**(ELISA - not as simple as we thought)**

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

## Evolution of pneumococcal ELISAs

** **

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

## Reverse Cumulative Distributions of Pooled Post Dose 3 ELISA Antibody Conc. for 7 Serotypes (Aggregate Data from 3 Studies) in Infants

- 0.35

- Pooled VE: 93.0%

- Predicted VE with 0.35 g/mLcutoff : 92.5%

**0.35**

- Immunized unweighted
- pooling

- Predicted VE

- Unimmunized
- Unweighted pooling

- Data from Dr. Kohberger, WHO 2003

***North CA KP**
** ****Navajo**
** ****South Africa**

## Future Direction

***Streptococcus pneumoniae***** polysaccharides**
**covalently bound to individual beads**

**Future Direction**
**Multiplex “ELISA” using Luminex System**
**(automated with high throughput) **

**Pickering, et al. 2002**

## Opsonophagocytic Assay

**(functional antibody)**

** ****Reference method, ****killing**** assay (Romero-Steiner, 1997) ****	****-**** ****Multilab study submitted for publication**** ****	****- Protocol  **_[www.vaccine.UAB.edu](http://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)**

## 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    **

## Correlation of ELISA and OPA (North CA KP infant study)

- ( p < 0.0001)

-   R = 0.80
- ( p < 0.0001)

- 0.01

- 0.1

- 1

- 10

** ****ELISA Concentration (********g/ml)**

- 1

- 10

- 100

- 1000

- 10000

** ****OPA TITER**

**7VPnC**

**Control**

-   **R = 0.92**
**( p < 0.0001)**

**Total N = 79**

**ELISA concentration of 0.20 ******** OPA titer of 1:8**

**Jodar, et al. 2003. Vaccine**** **

**Correlation of ELISA and OPA (North CA KP ****infant**** study)**

## Opsonic GMTs for young and old adults vaccinated

**Serotype**

**Opsonophagocytic GMT**

**Young**

**63-79 Yr**

**Opsonic GMTs for young and old adults vaccinated**
**once with a ****polysaccharide**** vaccine**

**Romero-Steiner, 1999. CID, 29:281**

## ...........................................................................                                                               

**GMT (opsonic titer)**

**GMC IgG ELISA (ug/ml)**

**   ****Pre**
**Post 7V #1**
**Post 7V #2**
**Post 23V**
**   ****Pre**
**Post 23V**

**Serotype 23F**

**Opa**

**Opa**

**Opa**

**Opa**

**Opa**

**Opa**

**Opa**

**ELISA**

**ELISA**

**ELISA**

**ELISA**

**ELISA**

**ELISA**

**ELISA**

**Serotype 19F**

**Serotype 18C**

**Serotype 14**

**Serotype 9V**

**Serotype 6B**

**Serotype 4**

- Vernacchio, et al. 2000. JID 181.

- ...........................................................................

- ...........................................................................

- ..............................

## Slide 13

## 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***** < .05) **

**Memory in Infants**

**Wuorimaa, Vaccine, 2001:19**

## Young adult antibody response after one dose of conjugate

**Months**

**Antibody Level (ug/ml)**

**Young adult antibody response after ****one dose**** of conjugate**
**and ****boosted**** with polysaccharide vaccine**

**CV**

**Ps**

**Powers, 1996. JID 173:1014**

**Wyeth-Lederle vaccine**

## Young adult antibody response after two doses of conjugate

**Months**

**Antibody Level (ug/ml)**

**Young adult antibody response after two**** doses**** of conjugate**
**and ****boosted**** with polysaccharide vaccine**

**CV**

**Ps**

**Kroon,  2001. Vaccine, 19:886**

**CV**

**Merck  vaccine**

## 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**** **

## 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, 2001:19**

## Avidities After a Single Dose of Conjugate Vaccine

**Avidities After a ****Single**** Dose of Conjugate Vaccine**
** ****in Adults**

**Avidity Index (%)**

**   ****Ps 1        Ps 5       Ps 6B    Ps 14      Ps 19F    Ps 23F**

**0 days**

**28 days**

**14 days**

**Extracted from Wuorimaa, Vaccine, 2001:19**

## DAYS Postvaccination

**Avidity Index (%)**

**Wyeth-Lederle vaccine**

**Extracted from Wuorimaa, Vaccine, 2001:19**

***Alum gave slight increase**

**DAYS Postvaccination**

## 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.**