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1.
The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18.4 (+/-4.2) and selective use of IGHD6 (P < 0.001) and IGHJ6 (P < 0.01) family genes. The clones expressing IGHV1-69 and IGHV 3-21 provided significant passive protection against invasive pneumococcal disease in vivo.  相似文献   

2.
Some anti-phosphocholine antibodies protect mice against challenge with certain, but not all, pneumococcal types. We found that both their isotype and reactivity with the cell wall C-polysaccharide of encapsulated pneumococci, as measured by immunodiffusion, were important in predicting the protective activity of anti-phosphocholine antibodies. We propose that the specificity of the protective antibodies includes the backbone of the phosphocholine-containing structure.  相似文献   

3.
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.  相似文献   

4.
The generation of antibodies is one of the first requirements in the characterisation of a newly cloned protein. However, this requires expression and purification of the protein in sufficient yield and purity for immunisation of animals and screening of fusion wells. Even with the development of highly efficient protocols based upon incorporation of specific peptide tags, this can be a tedious and time-consuming process. In an effort to improve the speed and efficiency of obtaining antibodies reactive with newly cloned proteins we have developed an approach based upon the expression of the protein at high level in cell lines originating from the species to be used for immunisation. To illustrate this approach we describe the generation of antibodies against two recently cloned proteins that are normally expressed at the membrane, the rat and mouse analogues of human decay accelerating factor (DAF; CD55). However, the strategy is applicable not only to membrane proteins but also to other proteins which can be expressed on the cell membrane by incorporating at the carboxy terminus the signal sequence for glycosyl phosphatidylinositol (GPI) anchor addition derived from DAF or another GPI-anchored protein. The strategy also permits rapid and efficient screening using flow cytometry on expressing cells.  相似文献   

5.
Antibodies are a key element of the immune response. They can bind their molecular targets with exquisite sensitivity and specificity, providing protection against a multitude of pathogens. They have long been understood to be markers of a successful response to vaccination, and are now widely manufactured as highly specific and robust immunotherapeutic agents. Less well understood are the polyreactive antibodies, found in serum, which are able to bind more than one target molecule. Here, we highlight new research into these naturally occurring polyreactive antibodies, which demonstrates their importance for protection against Streptococcus pneumoniae, a common cause of airway infection.

Antibodies against the xeno‐carbohydrate terminal galactose‐a‐1‐3‐galactose (anti‐αGal) are found in human plasma. These antibodies are common, constituting approximately 1% of immunoglobulins, and are found as IgG, IgM and IgA [1]. They may be generated in response to the carbohydrates expressed by intestinal bacteria [2]. The levels of these anti‐αGal antibodies, which can be of any isotype, are highly variable between individuals but may comprise up to 1% of total IgG and >1% of total IgM. The role of anti‐αGal antibodies in protecting against bacterial pathogens is unclear. While IgG antibodies are known to be particularly important in protective responses in the airways, the contribution of anti‐αGal antibodies to this protection has not previously been explored.Here, we present work [3] that reveals the importance of anti‐αGal antibodies in the protective immune response against Streptococcus pneumoniae, a leading cause of pneumonia, which results in millions of worldwide deaths annually. Antibodies recognizing the highly diverse structural components of the bacterial capsule are important for protection against disease, and the authors had previously reported evidence that anti‐αGal antibodies could react with pneumococci that did not contain the terminal Galα3Gal moiety for which the anti‐αGal antibodies are specific. This indicated that polyclonal human anti‐αGal antibodies could recognize a broader spectrum of bacterial antigens. Such ‘polyreactive’ antibodies have previously been proposed to be important in antibacterial immune responses [4]. Bernth Jensen et al. therefore aimed to understand the mechanisms by which anti‐αGal antibodies can display broad reactivity, and whether these antibodies contribute to protection against pneumococcal infection.Initial comparisons between healthy controls and patients with airway infections revealed decreased anti‐αGal levels in individuals with recurrent lower airway infections, even when corrected for age and blood group antigen—which are known confounders. Analysis of data from candidates for lung transplantation, who frequently experience lower airway infections, revealed that this group had increased anti‐αGal levels. Therefore, reduced anti‐αGal appears not to be a consequence of repeated lower airway infections, but may contribute to allowing such infections.To understand the specificity of anti‐αGal IgG, the authors examined reactivity against 91 S. pneumoniae serotypes, identifying positive reactions in at least 48. Of these, 37 are known not to contain the terminal Galα3Gal for which anti‐αGal displays its primary specificity. Carefully controlled follow‐up experiments confirmed that the anti‐αGal IgG contains reactivity to a range of bacterial polysaccharides, and is inhibited by the addition of soluble Galα3Gal; anti‐αGal can therefore be defined as polyreactive. Anti‐αGal antibodies provide an important contribution to the total antipneumococcal reactivity in individual samples, but this contribution is highly variable between individuals, and individuals’ anti‐αGal IgG is variable in the pneumococcal strains it binds. Furthermore, each individual''s anti‐αGal pool contains a wide range of specificities and should therefore be described as the plural ‘anti‐αGals’ or ‘anti‐αGal antibodies’.But do these polyreactive antibodies play an important contribution in protecting humans from pneumococcal disease? These authors had previously demonstrated that anti‐αGal antibodies were able to activate complement as is normal for IgG antibodies [5]. Here, they showed, in vitro, that anti‐αGal antibodies dramatically increased the phagocytic activity of primary human blood leucocytes, again by a complement‐dependent pathway. To understand whether they might also protect the population from disease, they analysed data from 29 034 reported cases of this notifiable infection in Denmark, between 1966 and 2014. From these data, they identified that frequently pathogenic subtypes reacted poorly with anti‐αGal, while the most anti‐αGal reactive pneumococcal subtypes caused invasive disease more rarely. Thus, these polyclonal anti‐αGal antibodies may protect the human population from invasive pneumococcal infections.This is an important study, revealing protective functions for polyreactive antibodies in the human population. Polyreactivity is often deemed an unsuitable quality during the development of antibodies for therapeutic purposes [6, 7], due to the possibility that off‐target specificities may reduce the therapeutic effect or generate undesirable effects. As this work indicates, a better understanding of how polyreactive antibodies provide protection against antigenically diverse pathogens may lead to improved strategies for preventing infection or limiting damaging autoimmune pathology.  相似文献   

6.
The availability of mouse monoclonal antibodies has been integral to the classification of human leukocyte cell surface proteins under the "Cluster of Differentiation" or "CD" nomenclature system. The sequencing of the human genome has identified many more proteins that have characteristics similar to the known leukocyte cell surface proteins, but which have not so far been identified using monoclonal antibodies. One factor that may have limited the generation of monoclonal antibodies to some of these proteins is the high level of sequence conservation between the mouse and human proteins, in particular in the extracellular regions that are recognized by most of the widely used antibodies. An alternative approach is to use a more distant species, such as chickens, for the generation of antibody reagents. Here we compare the extent of amino acid differences in the protein CD molecules expressed by human leukocytes and their mouse and chicken homologs. The analysis confirms that the human proteins are more similar to the mouse homologs than the chicken homologs. The results indicate that chicken antibodies have the potential to be used as an alternative to mouse reagents where human-mouse sequence conservation is high.  相似文献   

7.
Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.  相似文献   

8.
A number of studies have shown that the ratio of IgA1 and IgA2 subclasses in secretions can depend upon the nature of the antigen inducing their production. In order to evaluate the effect of the nature of the antigen on the subclass distribution of the naturally occurring salivary IgA antibodies against Streptococcus pneumoniae, we used enzyme immunoassay to measure the levels of natural IgA, IgA1 and IgA2 antibodies to pneumococcal capsular polysaccharide type 14 (PS14) and pneumococcal surface adhesin A (PsaA) in saliva of children during their first 2 years of life. The sum of anti-PS14 and anti-PsaA IgA1 and IgA2 correlated significantly with the antigen-specific total IgA, which showed that IgA1 and IgA2 add up to IgA. IgA1 was the predominant subclass for both antigens. The median of anti-PS14 and anti-PsaA IgA1 was higher than that of IgA2, and the antigen-specific IgA1 was found in a larger proportion of samples than IgA2. The ratio of IgA1 to IgA2 (IgA1/IgA2 ratio) was lower for anti-PS14 than for anti-PsaA, suggesting that the PS antigen induced more IgA2 than the protein antigen. The possible impact of the IgA subclass distribution on protection of mucosal surfaces by natural or vaccine-induced antibodies needs to be determined.  相似文献   

9.
Although the polysaccharide capsule of Streptococcus pneumoniae has been recognized as a sine qua non of virulence, much recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The individual contributions of pneumolysin (Ply), the major neuraminidase (NanA), autolysin (LytA), hyaluronidase (Hyl), pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA) have been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and comparing the impact on virulence in a mouse intraperitoneal challenge model. Mutagenesis of either the ply, lytA, or pspA gene in S. pneumoniae D39 significantly reduced virulence, relative to that of the wild-type strain, indicating that the respective gene products contribute to pathogenesis. On the other hand, mutations in nanA, hyl, or cbpA had no significant impact. The virulence of D39 derivatives carrying a ply deletion mutation as well as an insertion-duplication mutation in one of the other genes was also examined. Mutagenesis of either nanA or lytA did not result in an additional attenuation of virulence in the ply deletion background. However, significant additive attenuation in virulence was observed for the strains with ply-hyl, ply-pspA, and ply-cbpA double mutations.  相似文献   

10.
Monoclonal antibodies (mAbs) were generated against porins, one of the major outer membrane proteins of Salmonella typhi. Six clones, designated MP1, MP2, MP3 (IgG2ak), MPN4, MPN6 (IgG1k) and MPN5 (IgG2bk) were characterized by enzyme immunoassay (ELISA) for their reactivity to porins from S. typhi, Salmonella paratyphi A, S. paratyphi B, S. paratyphi C, Salmonella choleraesuis, Salmonella enteritidis, Salmonella krefeld, Salmonella panama, Salmonella typhimurium, Escherichia coli B, Shigella flexneri 1b and Pseudomonas aeruginosa. All the clones positive for S. typhi porins showed varying reactivity towards several Salmonella species. However, none of them was positive for porins from other Gram-negative bacteria or for lipopolysaccharide (LPS). The affinity constant of these mAbs, except MPN4, was found to be in the higher range. Dot ELISA revealed that the mAbs recognized porins only in their native form. The results of inhibition ELISA using horseradish peroxidase (HRP)-conjugated MP1 suggest that the clones MP1, MP2, MP3, MPN5 and MPN6 secreted antibodies to identical epitope(s) of a 36-kDa peptide and MPN4 to a different epitope of a 35-kDa peptide. The possible applications of these mAbs were discussed.  相似文献   

11.
The United Kingdom introduced meningococcal serogroup C conjugate (MCC) vaccines in 1999, resulting in substantial declines in serogroup C disease and carriage. Here, we measured the age-specific prevalence of serum bactericidal antibodies (SBA) to Neisseria meningitidis serogroup C and immunoglobulin G (IgG) concentrations to serogroups A, C, W-135, and Y in 2,673 serum samples collected in England between 2000 and 2004. We compared the seroprevalence of SBA titers of ≥8 in the postvaccination era with results from an earlier prevaccination study conducted using the same methods. We found that the percentages of individuals with protective SBA titers were higher in 2000 to 2004 in all of the age groups targeted for MCC vaccination. In the postvaccine era, the prevalence of protective titers was high (75%) in children who had recently been offered routine immunization, but this fell to 36% more than 18 months after scheduled immunization. In the cohorts targeted in the catch-up campaign, the percentage achieving SBA titers of ≥8 was higher in children offered the vaccine at ages 5 to 17 years than in children offered the vaccine at ages 1 to 4 years. The geometric mean concentration (GMC) IgG for serogroup C followed a similar pattern, corresponding to the age at and time since scheduled MCC vaccination. Serogroup-specific IgG GMCs for W-135 and Y were low and showed little variation by age. Serogroup A IgG GMCs were higher, possibly reflecting exposure to cross-reacting antigens. Although the incidence of serogroup C disease remains low due to persisting herd effects, population antibody levels to serogroup C meningococci should be monitored so that potentially susceptible age groups can be identified should herd immunity wane.  相似文献   

12.
Invasive infections caused by Streptococcus pneumoniae continue to be a major cause of morbidity and mortality worldwide, especially in children under 5 years of age. In the United States, 90% of invasive pneumococcal infections in children are caused by 13 serotypes of S. pneumoniae. The licensure (in 2000) and subsequent widespread use of a heptavalent pneumococcal conjugate vaccine (PCV7) have had a significant impact on decreasing the incidence of serious invasive pneumococcal disease (IPD) in all age groups, especially in children under 2 years of age. However, the emergence of replacement non-PCV7 serotypes, especially serotype 19A, has resulted in an increase in the incidence of serious and invasive infections. In 2010, a 13-valent PCV was licensed in the United States. However, the impact that this vaccine will have on IPD remains to be seen. The objectives of this review are to discuss the epidemiology of serious and invasive pneumococcal infections in the United States in the PCV era and to review some of the pneumococcal vaccines that are in development.  相似文献   

13.
14.
The aetiological diagnosis of community-acquired pneumonia (CAP) is challenging in children, and serological markers would be useful surrogates for epidemiological studies of pneumococcal CAP. We compared the use of anti-pneumolysin (Ply) antibody alone or with four additional pneumococcal surface proteins (PSPs) (pneumococcal histidine triad D (PhtD), pneumococcal histidine triad E (PhtE), LytB, and pneumococcal choline-binding protein A (PcpA)) as serological probes in children hospitalized with CAP. Recent pneumococcal exposure (positive blood culture for Streptococcus pneumoniae, Ply+ blood PCR finding, and PSP seroresponse) was predefined as supporting the diagnosis of presumed pneumococcal CAP (P-CAP). Twenty-three of 75 (31%) children with CAP (mean age 33.7 months) had a Ply+ PCR finding and/or a ≥2-fold increase of antibodies. Adding seroresponses to four PSPs identified 12 additional patients (35/75, 45%), increasing the sensitivity of the diagnosis of P-CAP from 0.44 (Ply alone) to 0.94. Convalescent anti-Ply and anti-PhtD antibody titres were significantly higher in P-CAP than in non P-CAP patients (446 vs. 169 ELISA Units (EU)/mL, p 0.031, and 189 vs. 66 EU/mL, p 0.044), confirming recent exposure. Acute anti-PcpA titres were three-fold lower (71 vs. 286 EU/mL, p <0.001) in P-CAP children. Regression analyses confirmed a low level of acute PcpA antibodies as the only independent predictor (p 0.002) of P-CAP. Novel PSPs facilitate the demonstration of recent pneumococcal exposure in CAP children. Low anti-PcpA antibody titres at admission distinguished children with P-CAP from those with CAP with a non-pneumococcal origin.  相似文献   

15.
We investigated pneumococcal carriage between children ≦5 years old with otitis media (OM) and those without. Non-PCV13 serotypes were common in both groups; 19A remained the second most common serotype among children with OM despite high PCV13 coverage. This is important when considering a schedule with reduced vaccine doses or reduced valency, and the modification of pneumococcal immunization schedule should be followed up closely to monitor the result of protection against pneumococcal infections.  相似文献   

16.
Nasopharyngeal carriage of Streptococcus pneumoniae is a key factor in the development of invasive disease and the spread of resistant strains within the community. A single nasopharyngeal swab was obtained from 648 unvaccinated children aged <5 years, either healthy or with acute respiratory tract infection or meningitis, during the winters of 2000 and 2001. The overall pneumococcal carriage rate was 35.8% (95% CI 32.1-39.6). The pneumococcal serotypes found most frequently in the nasopharynx were 14, 6B, 6A, 19F, 10A, 23F and 18C, which included five of the seven serotypes in the currently licensed seven-valent conjugate vaccine (PCV7); serotypes 4 and 9V were less common. Serotypes 1 and 5 were isolated rarely from the nasopharynx. A comparison of 222 nasopharyngeal isolates with 125 invasive isolates, matched for age and time to the carrier isolates, showed a similar prevalence of penicillin non-susceptible pneumococci (PNSp) (19.8% and 19.2%, respectively). PNSp serotypes were similar (6B, 14, 19F, 19 A, 23B and 23F) for carriage and invasive disease isolates. The coverage of PCV7 for carriage isolates (52.2%) and invasive isolates (62.4%) did not differ significantly (p 0.06); similarly, there was no significant difference in PCV7 coverage for carriage isolates (34.5%) and invasive isolates (28.2%) of PNSp. These data suggest that PCV7 has the potential to reduce pneumococcal carriage and the number of carriers of PNSp belonging to vaccine serotypes.  相似文献   

17.
The French Pediatric Infectious Diseases Group set up an active surveillance network to analyze the clinical and biological features of pneumococcal meningitis and the impact of the seven-valent pneumococcal conjugate vaccine (PCV7). From 2001 to 2005, 234 pediatric wards working with 166 microbiology laboratories enrolled all children with pneumococcal meningitis. Risk factors, signs and symptoms, vaccination status, cerebrospinal fluid analysis, treatments and case fatality rates were recorded. One hundred and sixty-nine centers (169/234) reported 616 cases, median age was 0.9 years and 67.2% of children were ≤2 years old. Underlying conditions were present in 13.1% of cases. The proportion of penicillin non-susceptible strains was 48.7%. Vancomycin plus a third-generation cephalosporin was prescribed in 92.7% of cases, and steroids were given before antibiotic treatment in 16.5% of cases. The case fatality rate was 10.8% overall and was not related to age, antibiotic susceptibility or steroid use. In children 2 to 24 months old compared to the prevaccinal period (2001–2002) a decrease of 28.4% of the number of cases was observed in 2005 (P < 0.05). Among children 2 to 24 months old, the proportion of serotypes covered by the PCV7 fell from 39/57 (68.4%) in 2001–2002 to 19/45 (42.2%) in 2005, while the proportion of non-vaccine serotypes and related serotypes increased respectively from 9/57 (15.8%) and 9/57 (15.8%) in 2001–2002 to 14/45 (31.1%) and 12/45 (26.7%) in 2005. Among 52 cases of pneumococcal meningitis that have occurred in vaccinated children (≥1 dose) with PCV7, 7 were due by vaccine serotypes. This study provides data on underlying conditions, penicillin susceptibility, serotype evolution according to vaccination status and risk factors for mortality for pneumococcal meningitis in children from 2001–2005 in France.  相似文献   

18.
An enzyme-linked immunosorbent assay (ELISA) for measuring antibodies against each of 14 polysaccharides in contemporary pneumococcal vaccine is described, and the findings of tests of paired sera from vaccinated human subjects are compared with those obtained by radioimmunoassay. The findings were in very poor agreement, and this appears to be due to the lesser ability of the ELISA procedure to measure antibody of low avidity. The ELISA procedure described here is not considered to be a satisfactory substitute for radioimmunoassay for measuring antibody responses to pneumococcal vaccine.  相似文献   

19.
Tetraspanins belong to a rapidly growing family of proteins characterized by the presence of four conserved transmembrane segments and are involved in such diverse functions as cellular activation, adhesion, migration and differentiation. In an effort to develop reagents against newly discovered tetraspanins, we have devised a simple method for the screening of monoclonal antibodies (mAbs) using recombinant GST fusion proteins. GST fusion proteins containing the second extracellular domain of different tetraspanins (CD9, CD63, CD53, CD81, A15 or CO-029) were produced separately. Mice were immunized with cells having a high expression of the chosen tetraspanin and the constructs were used to screen hybridomas in a solid phase ELISA. Several clones binding the fusion protein were identified for each construct tested: four anti-CD9 hybridoma clones, four anti-CD63, two anti-CD53, two anti-CD81, three anti-A15 and one anti-CO-029. All the newly developed mAbs recognized the native proteins by flow cytometry, immunofluorescence staining of cells and immunoprecipitation and bound to the denatured proteins on immunoblotting. Use of GST fusion protein constructs in a simple ELISA can facilitate screening for mAbs to members of the tetraspanin family, especially in cases where information is limited to the nucleotide sequence. The mAbs obtained by this strategy should prove to be valuable tools for functional studies of newly discovered tetraspanins.  相似文献   

20.
The process of T-lymphocyte differentiation within the thymus involves a series of molecular interactions. In this work we have carried out an analysis of the chick thymus microenvironment in order to evaluate its heterogeneity during development. We have produced 11 monoclonal antibodies whose staining patterns detected by the immunoperoxidase technique allowed us to divide them into five groups. A first group (E19-E2, P0-E5, and P15-T1) binds to thymic medullary stroma showing a reticular pattern on medullary epithelial cells and whose significance would be related to thymic stromal secretion. The second group of monoclonal antibodies (P15-T3) stains thymic corpuscles of 10- and 15-day chicks. The third group of antibodies includes P0-E1, P0-E3, P5-A6, and P15-T2 whose staining pattern is both medullary and cortical. The fourth group (P10-HB1 and P10-HB2) binds to thymic stromal and cortical thymocytes, and the fifth group (P5-A1) is characterized by the staining of medullary vessels of 5-day chicks. These five groups of monoclonal antibodies corroborate the existence of an antigenic diversity of the chick thymus microenvironment. Their possible relationships with T-cell differentiation and stromal-thymocyte interactions are discussed. © 1993 Wiley-Liss, Inc.  相似文献   

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