Asthma and antibodies to pneumococcal virulence proteins.

PURPOSE: We previously reported that asthmatics had lower anti-serotype-specific pneumococcal polysaccharide antibody levels than non-asthmatics, and the T-helper 2 (Th2) immune profile was associated with suboptimal pneumococcal polysaccharide antibody. Our objective was to determine the influence of asthma status on anti-pneumococcal protein antigen antibody levels. METHODS: We conducted a cross-sectional study, which enrolled 16 children and adults with asthma and 14 subjects without asthma. Asthma was ascertained by predetermined criteria. Serum IgG antibody levels to pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal choline-binding protein A (PcpA), and pneumolysin (PLY) were measured by enzyme-linked immunosorbent assays (ELISA). These antibody levels were compared between asthmatics and non-asthmatics. The Th2 immune profile was determined by IL-5 secretion from PBMCs cultured with house dust mite (HDM) and staphylococcal enterotoxin B (SEB) at day 7. The correlation between the anti-pneumococcal antibody levels and the Th2-HDM and SEB-responsive immune profile was assessed. RESULTS: Of the 30 subjects, 16 (53%) were male and the median age was 26 years. There were no significant differences in anti-PspA, anti-PspC, anti-PcpA, and anti-PLY antibody levels between asthmatics and non-asthmatics. The Th2 immune profile was inversely correlated with the anti-PspC antibody levels (r = -0.53, p = 0.003). This correlation was significantly modified by asthma status (r = -0.74, p = 0.001 for asthmatics vs. r = -0.06, p = 0.83 for non-asthmatics). Other pneumococcal protein antibodies were not correlated with the Th2 immune profile. CONCLUSION: No significant differences in the anti-pneumococcal protein antigen antibody levels between asthmatics and non-asthmatics were found. Asthma status is an important effect modifier determining the negative influence of the Th2 immune profile on anti-PspC antibody levels.

Clonal nature of the immune response. II. The effect of immunization on clonal commitment.

An inbred strain can produce several hundred different anti-group A carbohydrate (GAC) antibodies, as analyzed by isoelectric focusing. However, each individual mouse produces the bulk of its anti-GAC antibody as only one or two different spectrotypes, which appear to be randomly chosen. By using adoptive transfer techniques, we have observed that clonal commitment occurs very early in immunization, sometimes even before immunization, and thus does not result from competition among B cells for antigen. X

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

The IgM antibody response of mice to the streptococcal group A carbohydrate (GAC) was measured. With most strains tested, large amounts of IgM antibody were produced; in AKR mice, over 1% of the total nucleated spleen cells secreted IgM anti-GAC antibody after hyperimmunization. The relative avidity of the antibody was extimated by a modification of the Jerne plaque assay where spleen cells from individual mice were tested against erythrocytes with varying GAC epitope densitymthese studies showed that the earliest, as well as latest, IgM antibodies produced were highly restricted in avidity heterogeneity. No evidence of affinity maturation was seen upon hyperimmunization. These data favor the conclusion that the restricted IgG response seen in mice hyperimmunized to GAC is not the result of affinity driven competition for antigen among precursor cells.

The Salmonella typhimurium locus mviA regulates virulence in Itys but not Ityr mice: functional mviA results in avirulence; mutant (nonfunctional) mviA results in virulence.

The virulent Salmonella typhimurium strain WB600 carries the mviA allele of the gene mouse virulence A. As shown here, the virulent phenotype of WB600 is the result of a nonfunctional mviA gene. As compared to the functional allele mviA+, mviA increases virulence in Itys mice, but not in Ityr mice. A specific BglII site, mviA4185, between osmZ and galU, located at approximately 35 min on the salmonella chromosome, was within mviA. Insertion of an antibiotic cassette in the mviA4185 site of mviA+ or the homologous mviA4093 site of mviA DNA resulted in virulence when either cassette was recombined into the chromosome. When mviA and mviA+ were both expressed in the same strain with one carried in the chromosome and the other on a plasmid, avirulence was dominant. Replacement of the mviA allele of strain WB600 using P22 transductions of linked antibiotic cassettes cloned into the chromosome of virulent S. typhimurium strains (SR-11, TML, SL1344, C5, ATCC14028, W118-2, and WB600) showed that all but WB600 contained the avirulent mviA+ allele. Southern hybridizations provided no evidence for a second mviA allele anywhere in the genome of the six non-WB600 strains.

Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae.

In the mouse, most anti-PC antibody is found in one of the three murine anti-PC idiotype families: T15, M603, or M511. The antibodies within each of these idiotypic families have characteristic fine specificities for phosphorylcholine (PC)-analogues. In this paper we compare the ability of hybridoma IgM anti-PC antibodies of the three idiotype families to protect mice from fatal infection with S. pneumoniae. Antibody bearing the T15 idiotype was approximately 8 times as effective as antibody with the M603 idiotype and approximately 30 times as protective as antibody with the M511 idiotype. Reports by others have shown that the heavy chains of virtually all mouse anti-PC antibodies are produced by translocation of a single variable region gene and that the direct translation of this gene (in the absence of somatic mutations) results in heavy chains characteristic of the T15 idiotype. Thus, our findings suggest that the T15 germ line heavy chain variable region gene may have been selected through evolution to code for antibody binding PC-containing pathogens such as S. pneumoniae. Our observations may also explain the existence of regulatory mechanisms that result in maintenance of T15 idiotype expression in murine anti-PC immune responses.

Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA).

PspA is a cell surface protein of Streptococcus pneumoniae that is present on a number of clinical isolates as well as the nonencapsulated laboratory strain Rx1. In a previous report we have shown that mAbs directed against PspA can protect mice from at least some of the pneumococcal strains bearing this protein. In our present report we have produced insertional inactivation mutants that lack PspA and have used these mutants to demonstrate that PspA can play a role in pneumococcal virulence and that anti-PspA immunity can lead to protection against pneumococcal infection. PspA- mutants were obtained using derivatives of plasmid pVA891 carrying chromosomal fragments from Rx1. From one of the mutants, we cloned a 550 bp fragment of the pneumococcal gene into pVA891 and transferred this chimeric plasmid, designated pKSD300, into Escherichia coli. After transformation of pKSD300 into Rx1, PspA production is not detected. In colony hybridization experiments, the 550 bp fragment hybridizes specifically to pneumococcal isolates in a pattern consistent with the hypothesis that the fragment is a portion of the pspA structural gene that is different from the portions coding for the antigenic determinants detected by mAbs Xi64 or Xi126. When X-linked immunodeficient (xid) CBA/N mice were immunized with wild-type Rx1, they were resistant to challenge with type 3 strain WU2. However, when these mice were immunized with a PspA- mutant of Rx1, they failed to survive the subsequent challenge, indicating that immunity to PspA can contribute to the resistance to pneumococcal infection. Using pKSD300 we insertionally inactivated pspA in D39, a virulent strain of S. pneumoniae. When injected intravenously there was a 10-fold greater reduction of the mutant pneumococci in the blood, as compared to the wild-type D39.

Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae.

The antiphosphocholine (PC) antibody in normal mouse sera (NMS) provides protection against intravenous infection with encapsulated strain WU2 of type 3 Streptococcus pneumoniae. Mice unable to make anti-PC antibody, as a result of suppression with anti-T-15 idiotype or inheritance of the xid gene of CAB/N mice, are highly susceptible to infection with strain WU2. Mice inheriting the xid gene can be protected with NMS from immunologically normal mice or with IgM hybridoma anti-PC antibody. The protective effect of NMS can be removed with PC-containing immunoabsorbents.

Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guérin vaccines expressing pneumococcal surface protein A.

Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross-protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guérin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti-PspA immune sera elicited by the rBCG vaccines passively protected X-linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans.

Pneumococcal Vaccines.

Streptococcus pneumoniae is a Gram-Positive pathogen that is a major causative agent of pneumonia, otitis media, sepsis and meningitis across the world. The World Health Organization estimates that globally over 500,000 children are killed each year by this pathogen. Vaccines offer the best protection against S. pneumoniae infections. The current polysaccharide conjugate vaccines have been very effective in reducing rates of invasive pneumococcal disease caused by vaccine type strains. However, the effectiveness of these vaccines have been somewhat diminished by the increasing numbers of cases of invasive disease caused by non-vaccine type strains, a phenomenon known as serotype replacement. Since, there are currently at least 98 known serotypes of S. pneumoniae, it may become cumbersome and expensive to add many additional serotypes to the current 13-valent vaccine, to circumvent the effect of serotype replacement. Hence, alternative serotype independent strategies, such as vaccination with highly cross-reactive pneumococcal protein antigens, should continue to be investigated to address this problem. This chapter provides a comprehensive discussion of pneumococcal vaccines past and present, protein antigens that are currently under investigation as vaccine candidates, and other alternatives, such as the pneumococcal whole cell vaccine, that may be successful in reducing current rates of disease caused by S. pneumoniae.

Fusion proteins containing family 1 and family 2 PspA fragments elicit protection against Streptococcus pneumoniae that correlates with antibody-mediated enhancement of complement deposition.

PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.

Streptococcus pneumoniae: new tools for an old pathogen.

The pneumococcus is one of the longest-known pathogens. It has been instrumental to our understanding of biology in many ways, such as in the discovery of the Gram strain and the identification of nucleic acid as the hereditary material. Despite major advances in our understanding of pneumococcal pathogenesis, the need for vaccines and antibiotics to combat this pathogen is still vital. Genomics is beginning to uncover new virulence factors to advance this process, and it is enabling the development of DNA chip technology, which will permit the analysis of gene expression in specific tissues and in virulence regulatory circuits.

In vivo administration of bryostatin 1, a protein kinase C activator, decreases murine resistance to Salmonella typhimurium.

Bryostatin 1, a potent activator of protein kinase C, has antitumor activity against murine lymphoma, leukemia, and melanoma. In vitro, this compound stimulates the release of gamma-interferon, interleukins, and hematopoietic growth factors from accessory cells and activates both T- and B-cells. Bryostatin 1 is also able to stimulate neutrophils to undergo oxidative burst and degranulation. Because of the ability of this compound to stimulate the immune system, cause release of immune mediators, and activate neutrophils, we have examined its effect on bacterial infection by using the gram-negative bacterium Salmonella typhimurium in mice. We find that animals given injections i.v. of S. typhimurium have a shortened life span if they are also given injections i.p. of nonlethal doses of bryostatin 1. There is a dose-response relationship with 100 micrograms/kg bryostatin 1 having a greater effect on survival than 40 micrograms/kg. Below 40 micrograms/kg there are no effects on survival. Analysis of the first 4 h of Salmonella infection demonstrates that bryostatin 1 does not affect the blood clearance of the bacterium. However, by day 2 of infection greater numbers of bacteria are found in the livers and spleens of mice given injections of bryostatin 1. By day 5, 10-fold more S. typhimurium bacteria are found in the livers and spleens of mice receiving 40 micrograms/kg of bryostatin 1. To determine whether bryostatin 1 was affecting growth or causing the death of bacteria, we used a Salmonella carrying a plasmid which has a temperature-sensitive origin of replication and is unable to replicate when the bacteria are in mice. This experiment demonstrates that bryostatin 1 represses bacterial killing but does not affect bacterial growth. Bryostatin 1 given i.p. stimulates a transient syndrome of weight loss and diarrhea from which the mice recover and regain weight, suggesting that bryostatin 1 may release a number of important humoral mediators in vivo. The weight loss is exacerbated by Salmonella infection with mice receiving bryostatin 1 and S. typhimurium, in that they lose approximately 33% of body weight prior to death. Thus, at doses used to treat murine tumors, bryostatin 1 treatment does not affect the clearance of S. typhimurium from the blood but does decrease the killing of bacteria in the liver and spleen, leading to early animal death. Such potential effects of bryostatin 1 on the outcome of bacterial infections should be evaluated in ongoing human trials of this agent.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia 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.

Detection of isoelectric focused antibody by autoradiography and hemolysis of antigen-coated erythrocytes. A comparison of methods.

Mouse anti-group A streptococcal carbohydrate (GAC) antibodies were detected in analytical isoelectric focusing (IEF) acrylamide gels by the hemolysis of GAC-coated sheep erythrocytes immobilized in agarose. This procedure is rapid and reproducible but, because it requires diffusion of antibody from the acrylamide gel into the agarose layer, results in poorer resolution than other methods of detection where antibodies can be fixed in the acrylamide gel after focusing, such as protein staining of purified antibodies or autoradiography of 125I-GAC bound to focused antibodies.

Isoelectric focusing of immunoglobulins: improved methodology.

Conditions for isoelectric focusing and detecting antibodies in thin layers of polyacrylamide have been evaluated and several improvements have been made. First, we have developed a simple method for covalently attaching the polyacrylamide gel to the glass support which improves the mechanical stability of the gel and removes the need for protein subbed plates. This in turn leads to decreased electroendosmosis and decreased background protein staining. Secondly, we have applied the methods of Nguyen and Chrambach (1977 a,b), in which amino acid solutions are used as electrodes, to focus immunoglobulins. This has eliminated cathodic drift and resulted in extremely stable pH gradients. Finally, we have found that conventional methods for detecting focused antibodies that rely on protein cross-linking before exposure to antigen often lead to distortion of the focusing patterns. Both glutaraldehyde and suberimidate destroy the antigen binding capacity of some antibodies at concentrations too low for complete fixation of protein bands. These fixation artifacts are avoided if salt-precipitated antibodies are exposed to radiolabeled antigen before fixation.

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.

Systemic and mucosal protective immunity to pneumococcal surface protein A.

To date our studies demonstrate that PspA is a highly immunogenic molecule in mice and that it can elicit immunity to otherwise fatal infections following iv, ip, in, and it challenge. Although the molecule is serologically variable, it is sufficiently cross-reactive so that immunization with a single PspA can protect against strains of highly diverse serotypes. It is anticipated that a vaccine composed of a mixture of carefully chosen PspA molecules will be able to elicit protective immunity to virtually all pneumococci. If this vaccine proved efficacious in man, it would provide a more simple and less costly means of immunizing against pneumococcal infection than using recombinant vaccines. This could be especially important in the developing world where the cost of successful vaccines must be no more than pennies per dose. If PspA is found to be less efficacious than capsular polysaccharides, it may be valuable as a protein component of a PS-protein conjugate vaccine. In this capacity, PspA might expand the breath of protection elicited by a vaccine composed of only a few polysaccharide-protein conjugates representing capsule types most commonly associated with infectious pneumococci.