Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli.

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.

Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae.

Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity against Streptococcus pneumoniae it will be necessary to elicit protection against carriage. Capsular polysaccharide-protein conjugates, PspA, and PsaA are known to elicit some protection against nasopharyngeal carriage of pneumococci but do not always completely eliminate carriage. In this study, we observed that PsaA elicited better protection than did PspA against carriage. Pneumolysin elicited no protection against carriage. Immunization with a mixture of PsaA and PspA elicited the best protection against carriage. These results indicate that PspA and PsaA may be useful for the elicitation of herd immunity in humans. As PspA and pneumolysin are known to elicit immunity to bacteremia and pneumonia, their inclusion in a mucosal vaccine may enable such a vaccine to prevent invasive disease as well as carriage.

Characterization of a recombinant outer surface protein A (OspA) vaccine against Lyme disease.

Lyme disease, the most prevalent tick-borne disease in the United States, results from infection with Borrelia burgdorferi sensu stricto. Early studies of Borrelia burgdorferi sensu stricto identified outer surface protein A (OspA), a lipoprotein on the surface of spirochetes that could be the target of protective antibodies to this agent. Pasteur Mérieux Connaught has developed a Lyme vaccine, ImuLyme, using recombinant OspA protein (rOspA). Methods were developed to routinely assess the identity, quantity, structure, purity, biological activity, heterogeneity, stability, and potency of rOspA. In addition, several methods were performed on a series of lots to support the routine testing methods and further our understanding of the physicochemical characteristics of rOspA. These tests were electrospray mass spectrometry, circular dichroism, two-dimensional gel electrophoresis, amino acid analysis, peptide mapping with peptide sequencing, and the application of proteomic methodology to identify trace contaminant host cell proteins. The results of these methods indicate that the rOspA lots are composed of highly purified and properly processed and folded rOspA with trace amounts of E. coli host cell proteins.

Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.

Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA 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, Spodoptera frugiperda (Sf9) and Trichoplusia 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 approximately 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 approximately 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 microl, 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 S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.

Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi.

The lipoprotein outer surface protein A (OspA) of the Lyme disease agent. Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette. Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.

Carboxyl-terminal determinants of Abelson protein important for lymphoma induction.

The carboxyl-terminal region of the Abelson protein is not absolutely required for Abelson virus transformation. However, Abelson virus strains encoding proteins missing portions of this region have a reduced ability to transform lymphoid cells in vitro and in vivo. One such strain, called P90A, is unique in that P90A-injected mice almost always develop tumors containing highly oncogenic variants that encode new forms of Abelson protein. In this work, we have examined the mechanism by which these variants are generated and used the variants to identify carboxyl-terminal protein sequences important for the induction of Abelson disease. Analysis of mice injected with helper-free P90A virus stocks demonstrates that the variants are generated during viral replication in vivo, probably as a consequence of error-prone reverse transcription. The sequence of the P90A viral genome reveals that a 19-base deletion is responsible for synthesis of the truncated Abelson protein. As a consequence of this mutation, 167 carboxyl-terminal amino acids normally found in the wild-type protein have been replaced by 33 amino acids derived from an alternative reading frame. Site-directed mutants show that the combination of the deletion and the P90A carboxyl terminus is required for the generation of variants. Thus, the particular structure of the P90A protein, not the specific residues lost or gained, alters the transforming potential of the Abelson protein. Finally, the sequence of the variants encoding smaller Abelson proteins reveals that as few as 452 v-abl-encoded amino acids are required for rapid induction of Abelson disease.