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Baculovirus Expression, Purification and Evaluation of Recombinant Pneumococcal Surface Adhesin A of <i>Streptococcus pneumoniae</i>

1999; Karger Publishers; Volume: 67; Issue: 3 Linguagem: Inglês

10.1159/000028060

1423-0291

Barun K. De, Jacquelyn S. Sampson, Edwin W. Ades, Syd Johnson, Annie Stinson, Jennifer Crook, Jean A. Tharpe, Robert C. Huebner, G M Carlone,

Pneumonia and Respiratory Infections

Pneumococcal surface adhesin A (PsaA), with a molecular mass of ∼37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of <i>Streptococcus pneumoniae. S. pneumoniae</i> serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length <i>psaA</i> sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, <i>Spodoptera frugiperda</i> (Sf9) and <i>Trichoplusia</i> ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound ‘hydrophobic rPsaA’ (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of ∼38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed ‘soluble rPsaA’ (srPsaA) with a molecular mass of ∼37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 µl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with <i>S. pneumoniae</i> serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.