+------+---+--------------------------------------------------------------+
| **Ab |   | A third generation of Pnc vaccines is under development.     |
| stra |   | These vaccines are based on common proteins that are present |
| ct** |   | in all 90 known Pnc serotypes. The initial formulation of    |
|      |   | the vaccine contains two proteins: Pneumococcal Surface      |
|      |   | Adhesin A (PsaA) and Pneumococcal Surface Protein A (PspA).  |
|      |   | We intend to evaluate the epitopes in PsaA that are involved |
|      |   | in the adherence of Pnc to nasopharyngeal (NP) cells         |
|      |   | (Detroit 562 cells) and to examine how naturally induced     |
|      |   | antibodies to these epitopes influence Pnc adherence.        |
+------+---+--------------------------------------------------------------+
| **   |   | 1\. To determine the PsaA epitopes mediating the adherence   |
| Spec |   | of Pnc to NP epithelial cells. 2. To determine if Pnc        |
| ific |   | colonization or acute otitis media elicit antibodies to      |
| aim  |   | these functional epitopes.                                   |
| s:** |   |                                                              |
|      |   | Recently, a pneumococcal conjugate vaccine, Prevnar          |
| *    |   | (specific for the top 7 disease-causing serotypes) was       |
| *Bac |   | licensed in the United States for use in the pediatric       |
| k-** |   | population (Black, S. *et al.*). This vaccine is highly      |
|      |   | immunogenic and efficacious in children under 2 years of     |
| **g  |   | age. Since the introduction of the vaccine in the United     |
| roun |   | States, Active Bacterial Core surveillance data indicate     |
| d:** |   | that this vaccine has been efficacious in reducing disease   |
|      |   | in                                                           |
+------+---+--------------------------------------------------------------+
| *    |   | young children and also in adults that have not been         |
| *Bac |   | vaccinated (herd immunity, Whitney, C.G. *et al* ). However, |
| k-** |   | availability of this vaccine in other parts of the world is  |
|      |   | limited by its high cost, lower serotype coverage, and the   |
| **g  |   | lack of appropriate surveillance to justify the use of such  |
| roun |   | an expensive vaccine in countries with limited resources.    |
| d:** |   | This conjugate vaccine only covers 7 serotypes of the 90 Pnc |
|      |   | serotypes known. Therefore, a third generation of Pnc        |
| **   |   | vaccines is under development. These vaccines are based on   |
| Spec |   | common proteins that are present in all serotypes. The       |
| ific |   | initial formulation of the vaccine contains two proteins:    |
| des  |   | Pneumococcal Surface Adhesin A (PsaA) and Pneumococcal       |
| ign/ |   | Surface Protein A (PspA). PsaA is a putative Pnc adhesin and |
| Pla  |   | an ABC transporter for manganese. This multicomponent        |
| n:** |   | protein vaccine has undergone phase I trials with minimal    |
|      |   | adverse reactions in the adult human volunteers. Fold        |
|      |   | increases in IgG antibodies (no functional antibodies        |
|      |   | presented) to both proteins in pre-postvaccination sera were |
|      |   | found for the majority of study subjects (unpublished        |
|      |   | information presented at NVAC Sept.2003). The role of        |
|      |   | naturally developed antibodies to PsaA in prevention of      |
|      |   | colonization in humans has been previously demonstrated      |
|      |   | (Rapola, S. *et al*.). Anti-PsaA antibodies can reduce Pnc   |
|      |   | colonization and carriage in mice and protect chinchillas    |
|      |   | from otitis media (Briles, D.E. *et al.*). Other studies in  |
|      |   | mice have indicated that while antibodies to PsaA can        |
|      |   | prevent Pnc colonization and otitis media, antibodies to     |
|      |   | PspA can protect against pneumonia and invasive disease      |
|      |   | (Briles, D.E. *et al.* and Seo, J-Y *et al.*). *In vivo*,    |
|      |   | many factors contribute to pneumococcal (Pnc) colonization   |
|      |   | and multiple studies have also shown that adherence of Pnc   |
|      |   | to NP cells is highly mediated by the common cell-wall       |
|      |   | polysaccharide and choline-binding proteins (Cundell, D. R,  |
|      |   | *et al.*). More recently, we have shown that PsaA also       |
|      |   | mediates adherence to NP cells (Romero-Steiner, S. *et       |
|      |   | al.*). We intend to evaluate the epitopes in PsaA that are   |
|      |   | involved in the adherence of Pnc to nasopharyngeal cells     |
|      |   | (Detroit 562 cells) and determine how naturally induced      |
|      |   | antibodies to these epitopes influence Pnc adherence.        |
|      |   |                                                              |
|      |   | Characterization of the naturally occurring antibodies to    |
|      |   | PsaA is an important parameter in the evaluation of vaccine  |
|      |   | induced antibodies to this future Pnc vaccine. Availability  |
|      |   | of validated functional assays that can discriminate between |
|      |   | existing anti-PsaA antibodies and vaccine --induced          |
|      |   | antibodies is highly needed for vaccine licensure and future |
|      |   | evaluation of the immunogenicity of this protein vaccine.    |
|      |   | Preliminary studies indicate that sera from infants that     |
|      |   | have been recently Pnc colonized (positive NP aspirate) or   |
|      |   | with Pnc AOM (culture confirmed) have elevated antibodies to |
|      |   | PsaA in their sera. We intend to determine the PsaA epitopes |
|      |   | mediating the adherence of Pnc to NP epithelial cells and to |
|      |   | determine if Pnc colonization or acute otitis media elicits  |
|      |   | antibodies to these functional epitopes.                     |
|      |   |                                                              |
|      |   | **Selection of functional epitopes:** Three peptides have    |
|      |   | been previously selected by means of a phage display library |
|      |   | by Westerink, J.A. *et al.* We have located these peptides   |
|      |   | to the carboxy-terminus of the protein spanning regions that |
|      |   | have functional domains in the protein (Fig.1, unpublished). |
|      |   | These peptides are not highly homologous to the original     |
|      |   | amino acid sequence however they have the capacity to bind   |
|      |   | to nasopharyngeal epithelial cells and they protect against  |
|      |   | Pnc colonization in an animal model (Johnson, S. *et al*.).  |
+------+---+--------------------------------------------------------------+
| **S  | * |                                                              |
| tudy | * |                                                              |
| de   | F |                                                              |
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| Cont |   |                                                              |
| .)** | 1 |                                                              |
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|      | l |                                                              |
|      | a |                                                              |
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|      | h |                                                              |
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|      | d |                                                              |
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|      | a |                                                              |
|      | l |                                                              |
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|      | d |                                                              |
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|      | s |                                                              |
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|      | n |                                                              |
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|      | a |                                                              |
|      | f |                                                              |
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|      | z |                                                              |
|      | a |                                                              |
|      | t |                                                              |
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|      | h |                                                              |
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|      | c |                                                              |
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|      |   |                                                              |
+------+---+--------------------------------------------------------------+