Pneumococcal capsular polysaccharide preparations may contain non-C-polysaccharide contaminants that are immunogenic.

We measured the capacity to opsonize Streptococcus pneumoniae serotype 6B and estimated the concentration of immunoglobulin G anti-6B capsular polysaccharide (PS) antibodies in 25 pre- and postimmune sera from adults immunized with a pneumococcal PS vaccine. We first studied two postvaccination serum samples displaying less opsonophagocytic capacity than expected. The majority of anti-6B antibodies in the two samples reacted with the capsular PSs of several unrelated serotypes (2, 4, 9V, 19F, and 23F) and with the lysate of noncapsulated S. pneumoniae bacteria but not with C-PS. The non-type-specific antibodies accounted for at least one-half of anti-6B antibodies in 40% of prevaccination sera and 10% of postvaccination sera from adults. The non-type-specific antibodies could be demonstrated in the enzyme-linked immunosorbent assays (ELISAs) for pneumococcal antibodies to other serotypes (4, 9V, 18C, 19F, and 23F). The nonspecific antibodies appear to bind a contaminant(s) in the current preparations of capsular PS. ELISA for antibodies to pneumococcal capsules may not be serotype specific for some samples.     

Biological effects of anti-lipid and anti-protein monoclonal antibodies on Mycoplasma pneumoniae.

Monoclonal antibodies directed against Mycoplasma pneumoniae surface components were examined for their ability to block mycoplasma attachment to chicken erythrocytes. Purified preparations of antibodies which recognize the major mycoplasma ligand mediating cytadherence (protein P1, 165 kilodaltons) inhibited attachment by more than 85% of the control values. Monoclonal antibodies reactive with two other surface proteins of 110 and 32 kilodaltons also blocked attachment. Surprisingly, monoclonal antibodies specific for M. pneumoniae lipids (J. Morrison-Plummer, D. H. Jones, and J. B. Baseman, J. Immunol. Methods 64:165-178, 1983) enhanced mycoplasma-erythrocyte binding. All antibodies examined had no effect on thymidine incorporation by M. pneumoniae.     

Type-specific protection of neonatal rats from lethal group B streptococcal infection by immune sera obtained from human volunteers vaccinated with type III-specific polysaccharide.

Sera obtained from human volunteers at 6 weeks after vaccination with highly purified type III polysaccharide antigen prepared from a group B Streptococcus, strain M732, were found to protect neonatal rats from otherwise lethal infection by the homologous strain. The specific antibody content of the sera, expressed in micrograms of antibody protein per milliliter, was determined by an enzyme-linked immunosorbent assay in conjunction with quantitative precipitin analysis. For two sera studied in detail, the protective dose of antibody for 50% of the animals was 0.4 micrograms. Immune serum obtained from a volunteer who received type II polysaccharide vaccine was not protective against type III infection. Absorption of anti-type III serum by quantitative precipitation of antibodies with type III polysaccharide completely removed the passive protective activity of the serum. The results show that antibodies induced in humans by purified type II polysaccharide give serotype-specific protection in an animal model of neonatal infection.     

Plasma anti-pneumococcal antibody activity of the IgG class and subclasses in otitis prone children

Plasma anti-pneumococcal polysaccharide antibody activity (serotypes 3, 6a and 23) was determined in samples from 15 otitis prone children, at 30 months of age, and compared with age matched control children and adults. A recently developed enzyme immunoassay, using IgG subclass specific monoclonal antibodies for analysis of pneumococcal antibody activity of different IgG subclasses was employed. Adult sera always contained the highest antibody concentrations, almost exclusively of the IgG2 subclass. Children, on the other hand, had higher amounts of IgG1, significantly exceeding those of the adults, healthy children having higher IgG1, as well as IgG2 values than otitis prone children. The most significant differences were seen with type 6a, less so with type 23 antibodies. No differences in IgG1 anti-type 3 pneumococcal activity were observed between the children and the adults. These findings support the concept that pneumococcal antibody activity is confined to IgG2 and, in addition, it was found that sera from children contain antibodies of both IgG1 and IgG2 subclasses.     

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.     

Pneumococcal serotypes causing pneumonia with pleural effusion in pediatric patients

To determine the prevalence of serotypes of Streptococcus pneumoniae responsible for pneumonia with pleural effusion, we determined the capsular polysaccharide (PS) type directly on 49 pleural fluid specimens collected from pediatric patients during 2007 to 2009 with laboratory-confirmed pneumococcal pneumonia by using monoclonal antibodies and a multiplex, bead array immunoassay. Because the fluids had to be heated to remove nonspecific reactivity before being tested in the immunoassay and type 19A PS is heat labile, the pleural fluid samples were also tested for serotype 19A capsule gene locus by PCR. Use of the multiplex immunoassay combined with type-specific 19A PCR allowed for serotype determination on 40 of 49 pleural fluids. Pneumococcal pneumonia with pleural effusion was associated with a limited number of serotypes, with types 1, 3, 7F/A, and 19A accounting for 75% of the typeable cases. The concentration of capsular PS in the pleural fluids was often greater than 1 μg/ml and sufficient to inhibit the opsonic capacity of sera from individuals who had received the 23-valent pneumococcal PS vaccine. Based on the serotypes observed before and after introduction of the 7-valent pneumococcal conjugate vaccine, the recently licensed 13-valent pneumococcal conjugate vaccine may reduce the incidence of pneumonia with pleural effusions.     

Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae.

Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis.     

Evaluation of antibody responses to pneumococcal vaccines with ELISA and opsonophagocytic assay

Antibodies to a capsular polysaccharide (PS) provide protection against Streptococcus pneumoniae which express the homologous capsular serotype, and pneumococcal vaccines are designed to induce antibodies in the capsular PS. Levels and opsonophagocytic capacity of antibodies to the capsular PS of S. pneumoniae serotype 19F were determined by sera from adults immunized with 23-valent S. pneumoniae capsular PS vaccines. Geometric means of IgG anti-19F antibody level and specific opsonic titer rise significantly after immunization. The level of anticapsular PS antibodies for S. pneumoniae 19F serotype is fairly well correlated (r2=O.63) with the opsonophagocytic activities of sera. However, 3.7% (1/27) of serum samples display strikingly less opsonophagocytic activity than expected on the basis of their antibody level. Thus, antibody level may be of general use in predicting vaccine-induced protection among adults for 19F serotype. However, the opsonic activity data suggest that antibody levels are not always indicative of functional antibody.     

Rabbit antibodies to the cell wall polysaccharide of Streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci.

A conjugate, composed of the cell wall polysaccharide (C polysaccharide) of Streptococcus pneumoniae and bovine serum albumin (BSA), was prepared with the bifunctional agent N-succinimidyl-3-(2-pyridyldithio)-propionate. Analysis with monoclonal antibodies provided evidence that the phosphocholine (PC) moiety of the C polysaccharide was retained during the conjugation procedure. The C polysaccharide-BSA conjugate elicited antibodies to C polysaccharide in rabbits; no PC-specific antibodies were detected in globulins prepared from these hyperimmune sera obtained early and late after a second immunization. Rabbit hyperimmune sera were taken after multiple intravenous injections of the pneumococcus strain SRC-2, which has a capsulelike structure composed of the C polysaccharide. Globulin prepared from these antisera had both C polysaccharide- and PC-specific antibodies. Antibodies to C polysaccharide elicited by the C polysaccharide-BSA conjugate failed to protect mice against intraperitoneal challenge with a strain of type 3 or type 6A pneumococci. The anti-SRC-2 globulin conferred protection against both of these pneumococcal strains. Absorption of the SRC-2 globulin with C polysaccharide, however, failed to change its protective activity. These data provide evidence that antibodies to the C polysaccharide do not confer immunity against infection of mice with encapsulated pneumococci inoculated by the intraperitoneal route.     

Radioimmunoassay and opsonic antibody responses to pneumococcal capsular polysaccharide vaccine in serum and ascitic fluid of cirrhotic patients.

Pneumococcal capsular polysaccharide vaccine was administered to 19 cirrhotic patients and to 25 control subjects. Radioimmunoassay antibody concentration and opsonic titers (OT) were measured in sera and ascites collected before and 3 to 4 weeks after inoculation. The geometric mean antibody concentrations in prevaccination sera from the cirrhotic patients were significantly increased to types 3, 4, 7F, 8, 9N, and 12F antigens, and in postinoculation sera their geometric mean antibody concentration was increased to types 3, 9N, and 12F antigens. OT to Streptococcus pneumoniae type 3 correlated with the radioimmunoassay antibody concentration in postinoculation sera. Of 14 cirrhotic subjects, 3 had OT of greater than or equal to 4 in prevaccination sera, and the highest OT and radioimmunoassay antibody concentration were observed in postinoculation specimens from this group. Antibody and OT against S. pneumoniae type 3 were also observed in ascitic specimens. These data suggest that cirrhotic subjects respond to pneumococcal capsular polysaccharide with antibodies in both serum and ascitic fluid. However, the protective efficacy of this antibody response must be assessed by larger prospective studies.     

Antibody responses of splenectomized patients with non-Hodgkin's lymphoma to immunization with polyvalent pneumococcal vaccines.

The serum antibody responses of splenectomized patients with non-Hodgkin's lymphoma (NHL) who had been immunized with a polyvalent pneumococcal vaccine (Pneumovax 23) were evaluated by an enzyme-linked immunosorbent assay with the 23-valent pneumococcal vaccine as the antigen. A response to immunization, defined as a twofold-or-higher rise of the prevaccination titer of antibodies against Streptococcus pneumoniae polysaccharide, was elicited in 5 of 11 patients with NHL. No significant difference in the level of antibodies against S. pneumoniae polysaccharide between lymphoma patients and patients who had undergone splenectomy for other reasons was detected (P = 0.83 and 0.87 before and after vaccination, respectively). NHL patients who did not respond to the first immunization received a booster dose of the polysaccharide vaccine. This injection did not increase the pneumococcal-antibody titer significantly (P = 0.7). We conclude that vaccination with pneumococcal polysaccharides in splenectomized patients with NHL elicits an adequate antibody response in 45.4% of the cases and should therefore be administered. Revaccination of the nonresponders does not further increase the pneumococcal-antibody levels.     

Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S.

A human reference serum pool, lot 89-S, has been developed for use in quantitating concentrations of antibody to Streptococcus pneumoniae. Weight-based units have been assigned to antibodies to 11 pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) by using enzyme-linked immunosorbent assay methodology and a human standard reference serum, USNRP IS 1644. The experimentally derived assignments for anti-PnPs antibodies of the immunoglobulin G (IgG), IgM, and IgA isotypes in lot 89-S correlate well to the separately determined immunoglobulin assignment. These assignments for this antipneumococcal standard serum were used to quantitate IgG, IgM, and IgA isotype levels and the total immunoglobulin level in pediatric samples from a pneumococcal conjugate vaccine trial. The data indicate that these assignments may be used to assess levels of antibody to PnPs serotypes in human serum.     

Quantitative relationship between anticapsular antibody measured by enzyme-linked immunosorbent assay or radioimmunoassay and protection of mice against challenge with Streptococcus pneumoniae serotype 4.

We have recently shown that a substantial proportion of antibody to pneumococcal polysaccharide as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay is removed by adsorption with pneumococcal cell wall polysaccharide (CWPS). The present study was undertaken to validate the hypothesis that only serotype-specific antibody that remains after adsorption with CWPS provides protection against pneumococcal infection. Serum samples were obtained from human subjects before and after they had been vaccinated with pneumococcal polysaccharide vaccine. Antibody to Streptococcus pneumoniae serotype 4 was measured by ELISA without adsorption or after adsorption of serum with CWPS. Groups of mice were injected with graded doses of serum and then challenged intraperitoneally with 10, 100, or 1,000 50% lethal doses (LD50) of S. pneumoniae serotype 4. Without adsorption, prevaccination sera from five healthy adults appeared to contain up to 33 micrograms of antibody to S. pneumoniae serotype 4 antigen per ml; adsorption with CWPS removed all detectable antibody, and pretreating mice with up to 0.1 ml of these sera (less than or equal to 3.3 micrograms of antibody) failed to protect them against challenge with 100 LD50. In contrast, postvaccination sera contained 2.9 to 30 micrograms of antibody per ml that was not removed by adsorption. Diluting sera to administer desired amounts of serotype-specific immunoglobulin G showed a significant relationship between protection and antibody remaining after adsorption (P less than 0.05 by linear regression analysis); 150 ng was uniformly protective against 1,000 LD50, and 50 ng was protective against 100 LD50. These studies have, for the first time, quantitated the amount of serotype-specific antibody that protects mice against challenge with S. pneumoniae type 4. In light of these observations, it is necessary to reassess current concepts regarding the presence of antipneumococcal antibody in the unvaccinated population, responses to pneumococcal vaccination, and protective levels of immunoglobulin G.     

Diagnosis of pneumococcal pneumonia by enzyme-linked immunosorbent assay of antibodies to pneumococcal hemolysin (pneumolysin).

An enzyme-linked immunosorbent assay (ELISA) with a highly purified pneumolysin as the antigen was evaluated for serological diagnosis of pneumococcal pneumonia. One hundred four healthy controls were tested, and the specificity of the test was set to 95%. In samples from patients with bacteremic pneumococcal pneumonia, 82% (18 of 22) were positive, i.e., at least one serum sample had a titer above the upper normal limit or at least a twofold rise in antibody titers was noted. In nonbacteremic pneumococcal pneumonia, 45% (21 of 47) of samples were positive. All sera were negative for patients with pneumonia caused by Haemophilus influenzae, Legionella pneumophila, Chlamydia psittaci, and influenza A virus. However, in patients with a diagnosis of Mycoplasma pneumoniae infection, 8 of 25 (32%) samples were positive for antibodies to pneumolysin. All sera, including those from patients with mycoplasma infection, were negative to a protein control antigen by ELISA. Serum immunoglobulin G response to pneumolysin as measured by ELISA might thus be an aid in the laboratory diagnosis of pneumococcal pneumonia. This assay may also help to further elucidate the occurrence of dual infections with pneumococci.     

Correlation of serum opsonins with in vitro phagocytosis of Streptococcus pneumoniae.

C-reactive protein (CRP), an acute-phase reactant which binds to phosphocholine (PC) in the pneumococcal cell wall, and anti-PC antibodies are protective against experimental pneumococcal bacteremia in mice. To determine the relative opsonic capacities of CRP and anti-PC compared with those of antibodies against pneumococcal capsular polysaccharides (anti-PCP), we correlated in vitro opsonic activity for serotype 7F Streptococcus pneumoniae with concentrations of CRP, anti-PC, and anti-type 7F PCP in human sera from 10 normal subjects and 38 patients with sickle cell (SS) disease, a high-risk group for pneumococcal infection. Opsonic activity, measured by a radiolabeled bacterial uptake assay, correlated with anti-PCP levels but not with CRP or anti-PC in both the normal subjects and patients with SS disease. Addition of CRP to normal sera did not increase opsonic activity for serotypes 4 and 7F S. pneumoniae, although it did so for serotype 27, a nonpathogenic strain unique for having PC in its capsule. CRP and anti-PC were not effective opsonins when they bound to the pneumococcal cell wall rather than the capsule. The protective effects of CRP or anti-PC against these serotypes may be produced by means other than complement-dependent opsonization.     

Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG2 and/or IgG4

In order to define abnormalities of humoral immunity which determine susceptibility to respiratory tract infections in IgA-deficient adults, serum IgG subclass concentrations, and serum concentrations of pneumococcal antibodies and Haemophilus influenzae type B (Hib) antibodies sera from IgA-deficient adults with and without susceptibility to respiratory tract infections were compared. Infection susceptibility was not related to the degree of IgA deficiency, but was related to deficiency of IgG4 and, to a lesser extent, IgG2, as well as to low basal serum concentrations of pneumococcal polysaccharide antibodies. The combination of IgG2 and/or IgG4 deficiency and a non-protective basal serum concentration of antibody against two or more pneumococcal polysaccharides was present in the serum of six of 12 (50%) patients with severe infections, but only one of 44 (2%) patients without infections. Furthermore, the preservation of antibody responses against the most immunogenic pneumococcal polysaccharide type 3, but not against the less immunogenic types 7F, 9N and 14, in patients with severe infections suggested that abnormalities of pneumococcal polysaccharide antibody responses might include defects of affinity maturation.     

Prolonged and preferential production of polymeric immunoglobulin A in response to Streptococcus pneumoniae capsular polysaccharides.

Streptococcus pneumoniae is an invasive mucosal pathogen for which host defense is dependent on capsular polysaccharide-specific antibody. Capsule-specific immunoglobulin G (IgG), IgM, and IgA are produced following pneumococcal vaccination and infection. Serum IgA has two molecular forms, polymeric and monomeric. These forms may modulate the avidity of antigen binding and evolve over time as the immune response matures. Therefore, we sequentially characterized the molecular forms of serum IgA to three serotypes of pneumococcal capsular polysaccharides (types 8, 12F, and 14) after pneumococcal vaccination and after natural infection with type 14 S. pneumoniae. Although typically the form of IgA in antigen-specific systemic responses to protein antigens is predominantly polymeric in sera of patients shortly after exposure and shifts to the monomeric form in sera obtained several weeks later, the form of IgA in response to each pneumococcal capsular polysaccharide remained predominantly polymeric 1 month after natural infection and up to I year following vaccination. In contrast, IgA to pneumococcal cell wall polysaccharide was both polymeric and monomeric. Moreover, the form of IgA in response to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, was predominantly monomeric in the sera of 8 of 10 subjects tested 1 to 3 months after vaccination with either PRP alone or the diphtheria toxoid conjugate of PRP. We conclude that systemic responses to pneumococcal capsular polysaccharides are distinct in the production of predominantly polymeric IgA over time. The persistence of polymeric IgA may facilitate binding and clearance of pneumococci from the systemic circulation or reflect limited maturation of the immune response to pneumococcal capsular polysaccharides.     

Specificity of the antibody response to the pneumococcal polysaccharide and conjugate vaccines in human immunodeficiency virus-infected adults

Nonspecific antibodies, which are thought to be nonprotective, have been shown to contribute a substantial proportion of the measured concentration in the standardized immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for pneumococcal polysaccharide capsular antibodies. The presence of such antibodies in human immunodeficiency virus (HIV)-infected persons has not been evaluated. The amount of nonspecific antibodies is proportional to the reduction in IgG antibody concentration that occurs with serum absorption with the heterologous polysaccharide 22F. We measured the amount of nonspecific antibodies before and after vaccination with the pneumococcal conjugate vaccine (PCV; n = 33) or the pneumococcal polysaccharide vaccine (PPV; n = 34) in HIV-infected adults with CD4 counts of >/== 200 cells/mm3. Blood was drawn before and 2 months after vaccination. For prevaccination sera, we found a substantial amount of nonspecific antibodies for serotypes 4, 6B, 9V, and 23F (23 to 47% of measured IgG concentration), but not for serotype 14. There tended to be proportionately less nonspecific antibodies in postvaccine sera than prevaccine sera for PCV, but not for PPV. Subjects with a low HIV viral load (相似文献   

Enzyme immunoassay for detection of pneumococcal antigen in cerebrospinal fluid.

A solid-phase immunoassay utilizing horse antiserum against the C polysaccharide of Streptococcus pneumoniae and biotinylated rabbit antibodies to type-specific pneumococcal polysaccharides was developed to detect pneumococcal antigens in human body fluids and in broth cultures. Pneumococcal antigen could be detected in broth cultures of serotypes of S. pneumoniae containing as little as 10(2) to 10(3) organisms per ml. The assay system detected pneumococcal antigen in all 25 cerebrospinal fluid specimens obtained from patients with documented pneumococcal meningitis. There were no positive reactions noted in specimens from patients infected with Neisseria meningitidis group A or from patients without evidence of bacterial infection. The solid-phase enzyme immunoassay utilizing these reagents is a sensitive and specific assay for the immunodetection of a wide range of pneumococcal antigens.     

Production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci

Infections with Streptococcus pneumoniae remain a significant cause of morbidity and mortality. To gain insight into structure-function relationships for human antibodies to pneumococcal capsular polysaccharide (PPS), we studied the response of transgenic mice reconstituted with human immunoglobulin loci, XenoMouse, to PPS antigens in a pneumococcal vaccine. Enzyme-linked immunosorbent assays of sera from mice vaccinated with a 23-valent pneumococcal vaccine revealed that they produced serotype-specific human antibodies, with the greatest response being to the PPS of serotype 3 (PPS 3). Molecular sequence analysis of three monoclonal antibodies (MAbs) to PPS 3 generated from lymphoid cells from mice vaccinated with a 23-valent pneumococcal vaccine or a PPS 3-bovine serum albumin conjugate revealed that they all used heavy-chain immunoglobulin genes from the V(H)3 family, two expressed light chain genes from the human Vkappa1 family, and one expressed a mouse lambda light chain. The protective efficacy of the two MAbs was examined in mice. A 10-microgram dose of both, and a 1-microgram dose of one, significantly prolonged survival from a lethal serotype 3 infection in CBA/N mice. Our data show that XenoMouse mice produced protective, serotype-specific human antibodies to PPS 3, and they lend support to the proposal that these animals represent a useful model to study the human antibody response to PPS antigens.