Biomedical subjects

D E Briles

Publications and source records attributed to D E Briles.

At least 37 records · Page 2Linked to original sources

PubMed2000

Immunization of healthy adults with a single recombinant pneumococcal surface protein A (PspA) variant stimulates broadly cross-reactive antibodies to heterologous PspA molecules.

Pneumococcal surface protein A (PspA) is a highly variable protein found on all strains of pneumococci. To be successful, a PspA-based vaccine for S. pneumoniae must induce antibodies that are broadly cross-reactive. To address whether cross-reactive antibodies could be induced in man, we evaluated serum from adults immunized with recombinant clade 2 PspA from strain Rx1. Immunization with 5-125 microg rPspA lead to a significant increase in circulating anti-PspA antibodies, as well as antibodies reactive to heterologous rPspA molecules. Increased binding of post-immune sera to 37 pneumococcal strains expressing a variety of PspA and capsule types was observed, versus pre-immune sera. The extent of cross-clade reactivity of human anti-rPspA followed roughly the amount of sequence homology to the non-clade 2 antigens. It is hypothesized that priming of humans by natural exposure to S. pneumoniae contributes to the breadth of the cross-reactivity of antibody to PspA.

Adolescent↗

PubMed2000

The potential to use PspA and other pneumococcal proteins to elicit protection against pneumococcal infection.

Pneumococcal proteins, alone, in combination with each other, or in combination with capsular polysaccharide-protein conjugates may be useful pneumococcal vaccine components. Four proteins with a potential for use in vaccines are PspA, pneumolysin, PsaA, and PspC. In a mouse model of carriage, PsaA and PspC were the most efficacious vaccine proteins. Of these, PsaA was the best at eliciting protection against carriage. However, a combination of PspA and pneumolysin may elicit stronger immunity to pulmonary infection and possibly sepsis than either protein alone. Recently, a phase one trial of a recombinant family 1 PspA was completed in man. PspA was observed to be safe and immunogenic. Injection of 0.1 ml of immune serum diluted to 1/400 was able to protect mice from fatal infection with S. pneumoniae. Under these conditions, pre-immune serum was not protective. The immune human serum protected mice from infections with pneumococci expressing either of the major PspA families (1 and 2) and both of the pneumococcal capsular types tested: 3 and 6.

Adult↗

PubMed2000

Production and characterization of the functional fragment of pneumococcal surface protein A.

Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.

Amino Acid Sequence↗

PubMed2000

Human antibodies to pneumococcal surface protein A in health and disease.

BACKGROUND: Diseases caused by Streptococcus pneumoniae have a high impact in young children whose ability to mount antibodies to capsular polysaccharides is impaired. Pneumococcal surface protein A (PspA) is a potential vaccine candidate for this age group. METHODS: We used Western blot analysis and enzyme immunoassay to study human sera of healthy adults from Alabama (n = 20) and from Finland (n = 21), healthy children from Finland (n = 20) and ill children from Finland, those with pneumococcal invasive infection (n = 26) and those with nonpneumococcal invasive infection (n = 26). RESULTS: Human antibodies to PspA exhibited strong cross-reactivity among different pneumococcal strains. The geometric mean titer of IgG antibody to PspA in sera from 21 healthy adults was 4,040, from ten 3-year-old healthy children 1,080 and from ten 2-month-old healthy children 1,650. The geometric mean titer of PspA antibody of acute phase sera of children with invasive pneumococcal disease was 140, significantly (P < 0.001) lower than the respective value, 1,020, for children with infection caused by other bacteria. CONCLUSIONS: We demonstrate for the first time the existence of antibodies to PspA in human sera in health and disease. The findings in ill children suggest that antibodies to PspA might play a role in protection against pneumococcal disease.

Adult↗

PubMed2000

A latex bead-based flow cytometric immunoassay capable of simultaneous typing of multiple pneumococcal serotypes (Multibead assay).

A simple and rapid method of simultaneously determining 15 Streptococcus pneumoniae serotypes was developed. Fifteen latex beads of different sizes and different red fluorescence levels were coated with 1 of 15 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, and 23F) of pneumococcal capsular polysaccharide (PS). The bead mixture was incubated with individual pneumococcal lysate, a pool of rabbit antisera capable of binding the 15 serotypes, and fluorescein (green fluorescence)-conjugated anti-rabbit antibody. Bead size, red fluorescence, and green fluorescence were measured in a single flow cytometer run. The green fluorescence of the beads was inhibited only when there was a serotypic match between PS on the bead and PS in the pneumococcal lysate. This method distinguished cross-reactive serotypes and correctly identified the serotypes in 100% of 86 pneumococcal isolates tested.

Animals↗

PubMed2000

Diversity of PspA: mosaic genes and evidence for past recombination in Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a serologically variable protein of Streptococcus pneumoniae. Twenty-four diverse alleles of the pspA gene were sequenced to investigate the genetic basis for serologic diversity and to evaluate the potential of diversity to have an impact on PspA's use in human vaccination. The 24 pspA gene sequences from unrelated strains revealed two major allelic types, termed "families," subdivided into clades. A highly mosaic gene structure was observed in which individual mosaic sequence blocks in PspAs diverged from each other by over 20% in many cases. This level of divergence exceeds that observed for blocks in the penicillin-binding proteins of S. pneumoniae or in many cross-species comparisons of gene loci. Conversely, because the mosaic pattern is so complex, each pair of pspA genes also has numerous shared blocks, but the position of conserved blocks differs from gene pair to gene pair. A central region of pspA, important for eliciting protective antibodies, was found in six clades, which each diverge from the other clades by >20%. Sequence relationships among the 24 alleles analyzed over three windows were discordant, indicating that intragenic recombination has occurred within this locus. The extensive recombination which generated the mosaic pattern seen in the pspA locus suggests that natural selection has operated in the history of this gene locus and underscores the likelihood that PspA may be important in the interaction between the pneumococcus and its human host.

Alleles↗

PubMed2000

Acquired, but not innate, immune responses to Streptococcus pneumoniae are compromised by neutralization of CD40L.

Streptococcus pneumoniae is a significant pathogen of young children and the elderly. Systemic infection by pneumococci is a complex process involving several bacterial and host factors. We have investigated the role of CD40L in host defense against pneumococcal infection. Treatment of mice with MR-1 antibody (anti-CD154/CD40L) markedly reduced antibody responses to the pneumococcal protein PspA, elicited by immunization of purified protein or whole bacteria. In mice immunized with whole bacteria, MR-1 treatment reduced antibody responses to capsular polysaccharides but not cell wall polysaccharides. MR-1 did not suppress antibody responses to isolated capsular polysaccharides but did reduce the production of antibody to a capsular polysaccharide-protein conjugate, indicating that when presented in the context of whole bacteria, the humoral response to capsular polysaccharides is partially T-cell dependent. Despite the reduction of the protective humoral responses to pneumococcal infection, administration of MR-1 had no effect on sepsis, lung infection, or nasal carriage in nonimmune mice inoculated with virulent pneumococci. Thus, short-term neutralization of CD40L does not compromise innate host defenses against pneumococcal invasion.

Animals↗

PubMed2000

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.

Adhesins, Bacterial↗

PubMed2000

Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae.

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization-intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.

Adhesins, Bacterial↗

PubMed1999

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.

Animals↗

PubMed1999

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

Animals↗

PubMed1999

Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease.

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.

Amino Acid Sequence↗

PubMed1999

The pspC gene of Streptococcus pneumoniae encodes a polymorphic protein, PspC, which elicits cross-reactive antibodies to PspA and provides immunity to pneumococcal bacteremia.

PspC is one of three designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretory immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow et al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the pspA gene in S. pneumoniae and was thus called pspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many pspA alleles; clade B alleles are smaller and lack this pspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein. Immunization of a rabbit with a recombinant clade B PspC molecule produced antiserum that cross-reacted with both PspC and PspA from 15 pneumococcal isolates. The cross-reactive antibodies afforded cross-protection in a mouse model system. Mice immunized with PspC were protected against challenge with a strain that expressed PspA but not PspC. The PspA- and PspC-cross-reactive antibodies were directed to the proline-rich domain present in both molecules.

Amino Acid Sequence↗

PubMed1999

Tumor necrosis factor alpha receptor I is important for survival from Streptococcus pneumoniae infections.

Tumor necrosis factor alpha (TNF-alpha) is important in resistance to various microorganisms and provides signals to the target cells through two different receptors, TNF-alpha receptor I (TNFRI) (p55 receptor) and TNFRII (p75 receptor). To delineate the significance of the two different signaling pathways in resisting infections with extracellular bacteria, we examined the resistance of mice to Streptococcus pneumoniae (serotype 6B). TNF-alpha needs to be present early in infections, since one injection of wild-type mice with anti-TNF-alpha leads to an increased susceptibility of these mice to S. pneumoniae. TNF-alpha signaling through the p55 receptor (but not the p75 receptor) is crucial in resisting S. pneumoniae infections, because intraperitoneal injection of 100 CFU/mouse killed p55-deficient mice by day 2 of infection, whereas 1,000,000 CFU/mouse was needed to kill half of the control mice. p55-deficient mice do not show evidence of a deficient acute-phase response. All three types of mice (p55 deficient, p75 deficient, and normal) showed comparable rises in the levels of two acute-phase proteins (serum amyloid P and C3) at 24, 48, and 72 h after the experimental infections, and all of the mice showed comparable influxes of neutrophils to the site of infection. Finally, it was demonstrated that p55-deficient mice can be protected from the lethal effects of S. pneumoniae infection by injection of antibodies specific for S. pneumoniae polysaccharide capsule.

Acute-Phase Reaction↗

PubMed1999

Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the alpha chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

Animals↗

PubMed1998

A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A.

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.

Adjuvants, Immunologic↗

PubMed1998

The IS1167 insertion sequence is a phylogenetically informative marker among isolates of serotype 6B Streptococcus pneumoniae.

The phylogenetic utility of the IS1167 insertion sequence was examined with restriction fragment length polymorphism (RFLP) analyses of a sample of 50, predominantly invasive, capsular serotype 6B Streptococcus pneumoniae isolates previously characterized by multilocus enzyme electrophoresis (MLEE). The strains represented a genetically diverse assemblage of 34 distinct clonotypes composed of 26 restriction fragment types and 23 multilocus enzyme types. All isolates carried the IS1167 insertion sequence, with an average of 9.5 copies. The cross-classification of isolates based on RFLP and MLEE typing schemes was 81% concordant. Phylogenetic analyses demonstrated a significant (P < 0.0001) association between strains of a given RFLP lineage with those of a given MLEE lineage. A significant correlation (P < 0.00004) was also found between the proportion of restriction fragments shared by any given pair of isolates and their genetic distances estimated from the MLEE data. Parity between the two genetic markers implied that the sampled isolates were in linkage disequilibrium. The existence of nonrandom associations among genetic loci was confirmed by Monte Carlo analyses of the MLEE data. These studies, thus, demonstrated that invasive pneumococcal isolates of a single capsule type recovered on a regional scale can retain a largely clonal population structure over a period of 8 years. The ability to detect linkage disequilibrium and generate relatively congruent dendrograms based on distance and parsimony methods suggested that the restriction fragment data were robust to phylogenetic analysis.

Arctic Regions↗

PubMed1998

Pneumococcal diversity: considerations for new vaccine strategies with emphasis on pneumococcal surface protein A (PspA).

Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.

Animals↗