Isolation of a C (Ibc) protein from group B Streptococcus which elicits mouse protective antibody

The C (Ibc) proteins of group B Streptococcus (GBS) have been shown to induce mouse protective antibodies when present as immunogens on whole organisms. However, characterization of specific proteins responsible for inducing protection has not been reported. We have grown type Ic GBS in a dialysate of Todd Hewitt broth and analyzed the proteins extruded into the broth. Multiple proteins of varying size were visualized by SDS-PAGE. Ultrafiltration was used to separate the GBS components by molecular weight (MW) into 2 pools, those below 30,000 MW but above 10,000 MW (P10) and those above 30,000 MW (P30). The P10 contained 4 major proteins, including a 14,000 MW protein. Balb-c mice were immunized with the P10 fraction and the antisera used in mouse protection studies. This immune sera protected 100% of mice against challenge with type Ib GBS and protection was not altered by prior absorption of the sera with type Ia or Ib capsular polysaccharide. The P10 was fractionated by column chromatography and eluted proteins examined by SDS-PAGE and Western blot with the mouse protective antisera elicited to the P10. There was one major immunologically reactive protein at 14,000 MW which eluted in a partially purified form from the column. The 14,000 MW protein was reisolated from preparative SDS-PAGE gels and used to elicit antiserum in a rabbit. In mouse protection studies this rabbit antiserum protected mice against subsequent challenge with type Ib GBS (89% protection). Surface antigens were extracted from 125I-labelled type Ic GBS and immunoprecipitated with antiserum to the 14,000 MW protein. The 14,000 MW protein and multiple higher molecular weight proteins were immunologically cross-reactive suggesting the presence of shared epitopes. Thus the 14,000 MW protein from type Ic GBS that is antigenic and elicits mouse protective antibodies against the heterologous type Ib GBS fulfills the criteria for a C protein of GBS.     

Identification of group B streptococcal Sip protein, which elicits cross-protective immunity

A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6. 84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate.     

Variation in repeat number within the alpha C protein of group B streptococci alters antigenicity and protective epitopes.

Variable expression of repeating units of the protective alpha C proteins among clinical isolates of group B streptococci (GBS) may have implications for vaccine development. In this study, alpha C protein genes containing various numbers of repeats (1,2,9, and 16) were cloned in a T7 overexpression vector in Escherichia coli. Expression was induced by isopropyl-beta-D-thiogalactopyranoside, and proteins were purified by anion-exchange, gel filtration, or affinity chromatography or by isoelectric focusing. Rabbits were immunized with purified 1-,2-,9-, or 16-repeat proteins. All proteins appeared to be highly immunogenic. Enzyme-linked immunosorbent assay inhibition with 9-repeat protein as the coating antigen and 9-repeat-antigen-elicited antiserum showed that a 200-fold-higher concentration of 1-repeat antigen than of 9- or 16-repeat antigen was required for 50% inhibition of antibody-antigen binding. The concentration of 2-repeat antigen required for 50% inhibition was intermediate relative to the concentrations of 1- and 9-repeat antigens. These results suggested that antibodies to 9-repeat antigen recognized predominantly a conformational epitope(s) contained in proteins with higher numbers of repeats (9 or 16) but lost considerable binding affinities for an epitope(s) contained in alpha C proteins with fewer repeats (1 or 2). Similar results were obtained with antiserum to 16-repeat antigen. However, antibodies to 1- and 2-repeat antigens recognized 1-,2-,9-,and 16-repeat antigens with equal binding affinities. This finding suggested that 1- and 2-repeat-elicited antibodies recognized an epitope(s) on individual repeats. Loss of repeating units from the alpha C proteins may result in decreased protection because the loss of epitopes (including conformational epitopes) gives the microorganisms the opportunity to escape host antibodies. If 1- and 2-repeat-elicited antibodies bind all alpha C proteins with equal affinity, regardless of their repeat number, they may prevent GBS strains with fewer repeats from escaping host immunity. Protection data obtained with antisera to the proteins with different repeat numbers support this hypothesis: mouse pups challenged with GBS strain A909 were better protected when immunized with 1- or 2-repeat-elicited antiserum (76 and 75%, respectively) than when immunized with 9- or 16-repeat-elicited antiserum (41 and 48%, respectively).     

Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity.

Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes.     

Bacterial genetics and human immunity to group B streptococci

Serotype III group B Streptococcus agalactiae (GBS) are the most common cause of neonatal sepsis and meningitis. We have classified type III GBS by restriction digest patterns of chromosomal DNA and demonstrated that a subgroup of genetically related strains (RDP type III-3) causes the majority of type III GBS neonatal infection. Genetic differences between type III GBS strains contribute significantly to differences in virulence and host immune responses. While 100% of less virulent RDP type III-1 and III-2 organisms express C5a-ase, an inhibitor of neutrophil chemotaxis, only 63% of virulent RDP type III-3 isolates have functional C5a-ase. Functional differences in type III GBS C5a-ase are attributable to a shared genetic polymorphism, supporting our genetic classification. The mean capsular sialic acid content of virulent RDP type III-3 strains is significantly higher than that of less virulent strains, suggesting that capsular sialylation is also genetically regulated. C5a-ase is not critical for all RDP type III-3 strains to be invasive because the higher capsular sialic acid content of III-3 strains limits complement activation. The identification of these and additional genetic differences between GBS strains has important implications for our understanding of the pathogenesis of these important human infections.     

Streptococcal group B type antigen X in group L streptococci.

Extracts from 19 of 29 group L streptococcal cultures reacted distinctly with antiserum against group B streptococcal type antigen X in coagglutination and immunodiffusion tests. The X antigen corresponded to those obtained by streptococci of serological groups B and G.     

A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection.     

Isolation and properties of a novel IgG-binding protein from streptococci of serological group U

A nonimmune binding of immunoglobulin (Ig) G has been detected in streptococci of group U. The group U Fc-binding site differed from the five previously known types of staphylococcal and streptococcal Fc-binding sites by its strong affinity for murine IgG, with dissociation constants in nanomolar range for rat and mouse IgG, as well as for mouse IgG subclasses 1, 2a, 2b and 3. It also differed from other binding sites by the high sensitivity towards trypsin. The Fc-binding protein could be solubilized from the streptococci of group U with papain and purified by gel filtration on sephacryl S-200 and by subsequent affinity chromatography on human IgG-Sepharose. The purified binding protein was homogenenous on SDS-polyacrylamide gel electrophoresis and had a molecular weight of approximately 58,000 daltons. It retained its binding activities for murine IgG subclasses as revealed by western blotting. Coupled to CNBr-activated sepharose, the purified Fc-binding protein could be effectively used for the isolation of murine IgG subclasses by affinity chromatography.     

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.     

Prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity

The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.     

A monoclonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci.

Group B streptococci (GBS) are the leading causes of neonatal sepsis and meningitis in the United States, with a high rate of fatality and serious morbidity despite appropriate therapy. The C-protein antigens of GBS appear to be important in immunity to experimental infection, yet these antigens remain incompletely characterized with respect to their number, structure, and function. None of these proteins has yet been purified to homogeneity. We have developed a novel method for extraction of surface proteins from the A909 (Ia/c) strain of GBS by using mutanolysin. Antibodies raised in rabbits against these partially purified proteins conferred passive protection to lethal GBS infection in mice challenged with a GBS strain expressing C proteins with a heterologous capsule type. In addition, mouse monoclonal antibodies were produced and identified by reactivity with the mutanolysin-extracted proteins. One of these monoclonal antibodies (4G8) identifies an epitope on the alpha-antigen of the GBS C proteins (identified by protease susceptibility and mouse protection). On sodium dodecyl sulfate-polyacrylamide gels, this epitope appears as a series of regularly spaced bands ranging in apparent molecular mass from 160,000 to 30,000 Da. The monoclonal antibody 4G8 induces opsonic killing of GBS and protects mice from lethal challenge with GBS. Thus, the 4G8 monoclonal antibody identifies a fully protective epitope on the C-protein alpha-antigen of GBS.     

Characterization of an IgA receptor from group B streptococci: specificity for serum IgA

Some strains of group B streptococci express a cell surface protein which binds IgA. This report describes some properties of such an IgA receptor and compares it with a previously described IgA receptor from group A streptococci. The group B receptor was released in an almost pure form from bacteria incubated at elevated pH, and could be isolated by IgA-Sepharose affinity chromatography. The sequence of the N-terminal 19 amino acid residues was unique. The receptor preferentially binds IgA of human origin, as shown in immunoblotting experiments with purified IgA from nine different species. The affinity constant of the purified receptor for serum IgA was determined to be 3.5 x 10(8) M-1, but for secretory IgA it was too low to allow determination. This result indicates that secretory component and/or J chain interferes with the binding of IgA to this type of bacterial receptor, which may be one of the physiological functions of these polypeptides. A reduction in affinity was also observed for another complexed form of IgA, alpha 1-microglobulin-IgA. The group B receptor is antigenically unrelated to the IgA receptor from group A streptococci (protein Arp), but competitive inhibition experiments indicate that they bind to the same region in IgA. The implications of these findings, and the biological role of bacterial IgA receptors, are discussed.     

The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize

The ability to initiate organs throughout the lifecycle is a unique feature of plant development that is executed by groups of stem cells called meristems. The balance between stem cell proliferation and organ initiation is carefully regulated and ensures that organs can be initiated in regular geometric patterns. To understand how this regulation is achieved, we isolated a novel mutant of maize, fasciated ear2 (fea2), which causes a massive overproliferation of the ear inflorescence meristem and a more modest effect on floral meristem size and organ number. We cloned the fea2 gene using transposon tagging, and it encodes a membrane localized leucine-rich repeat receptor-like protein that is most closely related to CLAVATA2 from Arabidopsis. These findings provide evidence that the CLAVATA pathway for regulation of meristem size is functionally conserved throughout the angiosperms. A possible connection of fea2 to the control of crop yields is discussed.     

Characterization of the alpha antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody.

A murine monoclonal antibody raised against the alpha antigen of the group B streptococcal c proteins was analysed by immunofluorescence and whole-cell ELISA against a collection of 22 c protein-producing GBS. All the strains showing fluorescence and reactivity in ELISA turned out to be alpha antigen-carrying strains as defined by polyclonal rabbit antisera, while none of the strains producing only the beta antigen was positive. Western blot analyses of the alpha antigen released into the culture medium of growing bacteria suggest that the alpha antigen is present as distinct proteins of variable molecular weights. The upper limit of the molecular weights varies considerably from one strain to another, from approximately 200 kD to 70 kD. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD throughout the gel. Some strains gave rise to 15-16 bands, while others gave rise to only one or two bands. The present investigation suggests that alpha antigens include several, probably identical, repeating subunits of approximately 10 kD. The epitope recognized by the MAb seems to be located on a 10-kD fragment, and in addition, it appears to be surface located, making the MAb a suitable tool in serodiagnostic work.     

Species specific membrane anchoring of nyctalopin, a small leucine-rich repeat protein

Mutations in the gene NYX, which encodes nyctalopin, lead to the retinal disorder congenital stationary night blindness which is characterized by defective night vision (nyctalopia) from birth. Nyctalopin is of unknown function but is predicted to be a secreted glycoprotein of the extracellular small leucine-rich repeat (SLRP) proteoglycan and protein family attached to the cell membrane in humans via a glycosylphosphatidylinositol (GPI) anchor but in mouse via a transmembrane domain. We investigated membrane association and attachment for human and mouse nyctalopin and show, conclusively, that human nyctalopin is a GPI anchored protein. In addition, the orthologous mouse protein, although it localizes to the cell surface, is not GPI anchored. We also confirm both mouse and human nyctalopins are glycosylated. Further sequence analysis suggests that chimp, dog and frog nyctalopins are likely to be GPI anchored but that rat nyctalopin is not. This is the first reported example of orthologous proteins which have different mechanisms of cell membrane attachment. Notably, the disease-causing mutations that have been identified to date in the human NYX gene are all distributed throughout the core LRR region and not in the C-terminal GPI anchor signal sequence. We propose that the presence of nyctalopin on the surface of the cell rather than the mechanism of anchoring is crucial to its function.     

An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell-mediated immunity

Adjuvants are required to elicit protective immune responses with bacterial toxoids, inactivated viruses and subunit antigens produced by recombinant DNA technology. Some adjuvants, such as muramyl dipeptide (MDP) analog formulations, preferentially induce the formation of antibodies of isotypes that interact with complement and antibody-dependent effector cells, and do not elicit reaginic antibodies. Aluminum salts and mineral oil emulsions increase antibody formation but not cell-mediated immunity (CMI), whereas MDP formulations also elicit CMI. Adjuvants such as MDP and lipopolysaccharide (LPS) stimulate the production by accessory cells of IL-1 that increases the circulation of lymphocytes through draining lymph nodes and act as a growth factor for lymphocytes. Vehicles such as mineral oil emulsions, liposomes and Pluronic polymer formulations provide large surface areas on which antigens can be retained in a two-dimensional matrix, from which they can readily be transferred to antigen-presenting cells. The development of an adjuvant formulation able to elicit the formation of protective antibodies and CMI without unacceptable side effects is described.     

Characterization of neuraminidases produced by various serotypes of group B streptococci.

Neuraminidase produced by 11 strains of group B streptococci (GBS), from serotypes Ia, Ib, Ic, II, and III, were characterized according to molecular weight, antigenic identity, and substrate specificity. Following growth in a chemically defined medium, ammonium sulfate-concentrated culture supernatants were assayed for activity with bovine submaxillary mucin as substrate. Neuraminidase produced by GBS strain 122 (serotype III) was purified by a combination of salt fractionation, affinity chromatography with Affi-Gel Blue, ion-exchange chromatography with DEAE-cellulose, and gel filtration on Sephadex G-200. Purified neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of purified neuraminidase from strain 122 by 87.7%. The antiserum also reduced the activity of neuraminidases produced by the other four serotypes by between 78.3 and 90%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. The molecular weights obtained for the neuraminidases from the representative strains of each serotype ranged from 110,000 to 180,000. In addition, all of the GBS neuraminidases examined (regardless of the producing serotype) were active only on bovine submaxillary mucin. On the basis of these results, it appears that the neuraminidases produced by different GBS serotypes are quite similar.