Edwin Swiatlo

PspA, a protection-eliciting pneumococcal protein: immunogenicity of isolated native PspA in mice

PspA is a surface exposed virulence factor of S. pneumoniae that can elicit protective immunity to pneumococcal sepsis in mice. It can be released from pneumococci by washing them with a solution containing 2% choline chloride, by growing pneumococci in media containing 1.2% choline chloride, or by growing pneumococci in media in which the choline has been replaced by ethanolamine. Our results indicate that PspA is the major protection-eliciting antigen in each of these preparations. Two injections of < or = 1 microgram of native PspA purified by use of a choline-Sepharose column are highly immunogenic in BALB/c and CBA/N mice, and even in the absence of adjuvant can elicit protection against otherwise fatal sepsis with 100 times the LD50 of S. pneumoniae. Fragments comprising the N-terminal 115 and 245 amino acids of PspA were able to elicit protection but only in the presence of complete Freunds adjuvant (CFA). In the absence of CFA the 245 amino acid fragment was less than 1/100 as immunogenic as native PspA.

Molecular localization of variable and conserved regions of pspA and identification of additional pspA homologous sequences in Streptococcus pneumoniae

PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3 pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3 portion of pspA. A probe derived from the 5-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3 probes. Thus, the 3-half of pspA appears to contain more highly conserved sequences than the 5-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.

Pneumococcal Surface Protein A Is Expressed In Vivo, and Antibodies to PspA Are Effective for Therapy in a Murine Model of Pneumococcal Sepsis

ABSTRACT Pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Streptococcus pneumoniae (pneumococcus) and induces antibodies which protect against invasive infection in mice. Pneumococci used for infectious challenge in protection studies are typically collected from cultures grown in semisynthetic medium in vitro. The purpose of these studies is to confirm that PspA is expressed by pneumococci during growth in vivo at a level sufficient for antibodies to PspA to be protective. Mice were actively immunized with purified PspA or by passive transfer of monoclonal antibody (MAb) and challenged with a capsular type 3 strain in diluted whole blood from bacteremic mice. All were protected against challenge with 10 times the 50% lethal dose (LD50), and mice challenged with 1,000 times the LD50 had increased survival compared with controls. Additionally, nonimmune mice treated with MAbs to PspA or PspA immune serum at 6 and 12 h after infection with 10 times the LD50 also showed increased survival. Northern blot analysis of RNA from pneumococci grown either in vitro or in vivo showed similar levels of PspA mRNA. These results demonstrate that PspA is expressed in vivo in a mouse model and that immunization with PspA induces antibodies to an antigen which is expressed during the course of invasive infection. Immunotherapy with antibodies to PspA may have some utility in treating pneumococcal infections in humans.

Oligonucleotides identify conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is an immunogenic surface protein of Streptococcus pneumoniae. PspA of S. pneumoniae strain Rx1 is a 65-kDa protein composed of an alpha-helical N-terminus of 288 amino acids followed by an 82-amino-acid proline-rich region, 10 repeats of 20 amino acids each, and a 17-amino-acid C-terminus. It has been demonstrated that the 3-half of pspA is relatively conserved among unrelated pneumococcal isolates and the 5-half of the gene is highly variable. Additionally, nearly all pneumococcal strains contain at least one other locus with sequence homology to pspA. In this study oligonucleotides derived from the DNA sequence of pspA of Rx1 were used both as hybridization probes and as primers in the polymerase chain reaction (PCR) to investigate genetic variation within domains of pspA and in the pspA-like sequences from 18 strains representing 12 capsule and 9 PspA serotypes. Sequences encoding the leader peptide, the proline-rich region, and the repeat region are highly conserved among pspA and pspA-like sequences. The alpha-helical coding domain is highly diverse among pspA and pspA-like sequences of different strains.

Comparative in vitro activities of four new fluoroquinolones against Streptococcus pneumoniae determined by Etest

Clinafloxacin, levofloxacin, sparfloxacin and trovafloxacin were tested by Etest against 188 Streptococcus pneumoniae isolates. Clinafloxacin and trovafloxacin were 2-4-fold more potent than sparfloxacin and 8-fold more than levofloxacin. Two isolates, both serotype 6, with high-level quinolone resistance (> or = 8 micrograms/ml) were detected. The Etest is a practical means for determining S. pneumoniae susceptibilities to new fluoroquinolones.

COMPARATIVE ACTIVITIES OF PARENTERAL CEPHALOSPORINS AGAINST PENICILLIN-RESISTANT STREPTOCOCCUS PNEUMONIAE ISOLATED FROM PEDIATRIC PATIENTS

Abstract Thirty-eight isolates of penicillin-intermediate and 28 isolates of penicillin-resistant Streptococcus pneumoniae obtained from pediatric patients were tested against cefotaxime, ceftriaxone, cefuroxime, and ceftizoxime by microbroth dilution to determine minimum inhibitory concentrations (MICs). All penicillin-intermediate isolates were susceptible to ceftriaxone and cefotaxime, and the 90% MIC (MIC 90 ) for these drugs (0.5 μg/mL) was 2 dilutions less than those of cefuroxime and ceftizoxime (2 μg/mL). Of the penicillin-intermediate isolates, 71% were susceptible to cefuroxime. All drugs were significantly less active against penicillin-resistant strains with only 9 (32%), 8 (29%), and 0 (0%) of these strains susceptible to ceftriaxone, cefotaxime, and cefuroxime, respectively. For penicillin-resistant strains, MIC 90 was 4 μg/mL for ceftriaxone, 8 μg/mL for cefotaxime, 32 μg/mL for cefuroxime, and 64 μg/mL for ceftizoxime. This in vitro study demonstrated that ceftriaxone and cefotaxime are more active than cefuroxime and ceftizoxime against penicillin-intermediate and penicillin-resistant S pneumoniae isolated from children.

In vitro activities of cefepime versus cefotaxime and ceftriaxone against penicillin-resistant Streptococcus pneumoniae determined by Etest.

The Etest was used for determining in vitro susceptibilities of 144 unique clinical isolates of penicillin-intermediate and resistant Streptococcus pneumoniae to cefepime, cefotaxime, and ceftriaxone. MIC ranges were 0.12-8 mug/ml for cefepime and 0.06-16 mug/ml for cefotaxime and ceftriaxone. MICs for 50% of the isolates for the three agents were equivalent at 1 mug/ml, whereas MICs for 90% of the isolates were 2 mug/ml for cefotaxime and ceftriaxone, versus 4 mug/ml for cefepime. The Etest is a practical means for determining susceptibilities of S. pneumoniae to cefepime and other cephalosporins in diagnostic laboratories.