Multifunctionalized alpha,beta-cyclopentenones from C-2 and C-4-ulopyranosyl compounds: a stereospecific rearrangement initiated by base.

Base treatment of O-benzyl protected C-2- or C-4-ulopyranosyl compounds (4 alpha, 4 beta, and 11) by either 10% Et(3)N or 1% K(2)CO(3) in MeOH initiated a beta elimination to afford alpha,beta-unsaturated C-ulopyranosyl compounds (5 alpha, 5 beta, and 12), which further rearranged in a stereocontrolled manner to multifuctionalized alpha,beta-cyclopentenones (6 and 14) in 70-80% yield. Both C-alpha- and C-beta-2-ulosides (5 alpha and 5 beta) produced the same cyclopentenone 6, indicating that a 1,2-enolate is formed prior to the cleavage of the C-5--O bond. Because 6 is racemic, it was probably formed by the intramolecular cycloaldolization of two equally populated enantiomeric intermediates. When treated with 90% Et(3)N in MeOH, 5 alpha yielded almost exclusively 15 (isomer of 6), which was formed by a migration of the double bond in 5 alpha during the previously described rearrangement. Thus either 6 or 15 was the major product, depending on the base used.

Construction of multivalent sialyl Le(x) from the type Ia group B Streptococcus capsular polysaccharide.

The type Ia group B Streptococcus (GBSIa) capsular polysaccharide was specifically degraded by partial Smith oxidation of 2,3-diol of the Glc in the backbone to fragments representing asialo core repeating units. Sialylation of these oligomers furnished GBSIa multiple repeating units. One, two and three repeating units of GBSIa were obtained pure, and the higher oligomers were obtained as mixtures. After enzymatic fucosylation oligosaccharides carrying bivalent, trivalent and other multivalent sialyl Le(x) epitopes presented as appendages on an oligolactoside scaffold were obtained.

Immunostimulant oxidized beta-glucan conjugates.

beta-Glucans are polysaccharides that act as nonspecific immune system stimulants. However, many beta-Glucans are sparingly soluble in water. This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties. Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants. Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-[6-(N,N'-dimethylamino)purin-9-yl]pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages. In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess.

The conformational epitope of type III group B Streptococcus capsular polysaccharide.

In order to further characterize the conformational epitope of GBSPIII, we synthesized various oligosaccharides with the GBSPIII-related structures by a tailor-assembly synthetic scheme and a more traditional block-wise chemo-enzymatic approach. The oligosaccharides were used to probe the conformational epitope of GBSPIII using number of complementary techniques. The protective epitope of GPSPIII was further defined as length-dependent and conformational. The results of the studies confirmed that two repeating units (2RU) is the minimum binding unit and the epitope optimization mainly takes place between chain length 2RU to 7RU. Epitope optimization and multivalency were observed between 7RU and 20RU. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self-antigens, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide.

NMR and molecular modeling of complex carbohydrates and carbohydrate-protein interactions. Applications to anti-bacteria vaccines.

In order to characterize the conformational epitope of the group B meningococcal polysaccharide and of the type III group B Streptococcus capsular polysaccharide NMR measurements were done on a wide variety of native and modified polysaccharides and oligosaccharides. Since these saccharides are highly mobile and exist as random coils in solution, the analysis of the NMR data and molecular modeling was done to take into account this inherent flexibility. The conformational model of extended high-order helices being selected upon binding to a protein, although still hypothetical at this stage, has proven useful in explaining the serology for the conformational epitopes for polysaccharides of group B Neisseria meningitidis, group B Streptococcus type III and Streptococcus pneumoniae type 14.

Biochemical engineering of surface alpha 2-8 polysialic acid for immunotargeting tumor cells.

To target tumor cells for immunotherapy, we evaluated the feasibility of altering the epitopes on the surface polysialic acid of tumor cells. A precursor (N-propionylmannosamine), when incubated with leukemic cells, RBL-2H3 and RMA, resulted in substitution of the N-acetyl groups of surface alpha2-8 polysialic acid with N-propionyl groups. Expression of the altered alpha2-8 N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes alpha2-8 N-propionylated polysialic acid. The expression of alpha2-8 N-propionylated polysialic acid and the lysis of tumor cells by antibody-dependent cytotoxicity depended on the time and dose of incorporation of N-propionylated mannosamine. In vivo, mAb 13D9 effectively controlled metastasis of leukemic cells RMA when mice were administered the precursor N-propionylated mannosamine.

T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria.

Immunologic paradigms classify bacterial polysaccharides as T cell-independent antigens. However, these models fail to explain how zwitterionic polysaccharides (Zps) confer protection against intraabdominal abscess formation in a T cell-dependent manner. Here, we demonstrate that Zps elicit a potent CD4+ T cell response in vitro that requires available major histocompatibility complex class II molecules on antigen-presenting cells. Specific chemical modifications to Zps show that: 1) the activity is specific for carbohydrate structure, and 2) the proliferative response depends upon free amino and carboxyl groups on the repeating units of these polysaccharides. Peptides synthesized to mimic the zwitterionic charge motif associated with Zps also exhibited these biologic properties. Lysine-aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T cells in vitro and conferred protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus. Evidence for the biologic importance of T cell activation by these zwitterionic polymers was provided when human CD4+ T cells stimulated with these molecules in vitro and adoptively transferred to rats in vivo conferred protection against intraabdominal abscesses induced by viable bacterial challenge. These studies demonstrate that bacterial polysaccharides with a distinct charge motif activate T cells and that this activity confers immunity to a distinct pathologic response to bacterial infection.

Murine immune response to Neisseria meningitidis group C capsular polysaccharide: analysis of monoclonal antibodies generated in response to a thymus-independent antigen and a thymus-dependent toxoid conjugate vaccine.

Antibody (Ab) responses to polysaccharides (PSs) such as Neisseria meningitidis group C PS (MCPS) are characterized as being thymus independent (TI) and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled to tetanus toxoid (MCPS-TT), elicits a thymus-dependent (TD) response. In order to understand the influence of the form of a vaccine (TI versus TD) on the Ab repertoire, we generated monoclonal antibody (MAb) panels from mice immunized and boosted with MCPS or MCPS-TT in different ways. The panels of MAbs were examined for isotype, fine specificity, affinity, and V(H) gene family usage. The use of MCPS-TT resulted in a shift in the isotype from immunoglobulin M (IgM) and IgG3 elicited in response to the MCPS to primarily IgG1. This isotype shift was accompanied by a change in the fine specificity of the response to the conjugate compared to that of PS. New fine specificities and increased affinity were observed in response to the TD antigen (Ag). Dot blot and Northern analyses of MCPS MAbs revealed that V(H) gene family usage is dominated by V(H)J558, used by 23 of 39 MAbs. V(H)3609 was seen in three MAbs of restricted fine specificity. V(H)Q52, V(H)7183, and V(H)VGAM3-8 were seen in more than one MAb across these panels, while V(H)10 and V(H)X24 were detected only once in response to the TI-2 Ag. All MAbs in the panels utilized kappa light chains, and all functional J(kappa) genes were expressed.

Synthesis of a disialylated hexasaccharide of type VIII group B Streptococcus capsular polysaccharide.

As part of our program to design, develop and prepare protective vaccines against the bacterial pathogens Group B Streptococcus, we report the synthesis of a disialylated hexasaccharide. This hexasaccharide represents a portion of the serotype-specific capsular polysaccharide of Type VIII that has the tetrasaccharide repeat unit [beta-L-Rhap-(1-->4)-beta-D-Glcp-(1-->4)-[alpha-Neu5Ac-(2--> 3)]-beta-D- Galp-(1-->4)]n. A tetrasaccharide corresponding to this repeat unit has been synthesized by us [E. Eichler, H.J. Jennings, D.M. Whitfield, J. Carbohydr. Chem., 16 (1997) 385-411]. Since the protective epitopes are believed to involve several repeat units, methods to extend this tetrasaccharide were examined. This objective requires a glycosylation of the unreactive OH-4 of the beta-L-Rhap, which was accomplished by coupling a D-Galp glycosyl trichloroacetimidate donor with a beta-L-Rhap-(1-->4)-D-Glcp acceptor. Subsequent coupling of this trisaccharide as a donor to an alpha-Neu5Ac-(2-->3)-D-Galp disaccharide acceptor gave a pentasaccharide. The pentasaccharide was deprotected and enzymatically sialylated using an alpha-(2-->3)-sialyltransferase from Campylobacter jejuni to give the title hexasaccahride alpha-Neu5Ac-(2-->3)- beta-D-Galp-(1-->4)-beta-L-Rhap-(1-->4)-beta-D-Glcp-(1-->4)-[alpha -Neu5Ac- (2-->3)]-beta-D-Galp-(1-->O)-(CH2)3N3.

Conformational epitope of the type III group B Streptococcus capsular polysaccharide.

The protective epitope of the type III group B streptococcal polysaccharide (GBSPIII) is length dependent and conformational. To obtain a more accurate characterization of the conformational epitope, ELISA inhibition and surface plasmon resonance studies were conducted on two GBSPIII-specific mAbs using a large panel of oligosaccharide probes. The results of the studies confirmed that 2 repeating units (RU) is the minimum binding unit and that, while increases in chain length from 2 RU to 7 RU caused further optimization of the epitope, it remained monovalent. A 3-fold increase in affinity was observed between 7 RU and 20 RU, which, by surface plasmon resonance studies on a Fab, was shown to be due to both further optimization of the individual epitope and the occurrence of multivalency of epitope. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self Ags, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII, the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide. Contrary to a previously proposed model of GBSPIII binding in which the binding of the first Ab propagates a continuum of helical epitopes, our binding kinetics are consistent only with the helical epitope's being discontinuous and infrequent.

Synthesis and NMR assignments of galactosylgloboside and its beta-D-GalNAc-(1-->4)-alpha-D-Gal-linked positional isomer in a conjugatable form.

Two pentasaccharides suitable for conjugation, namely 3-aminopropyl glactosylgloboside and its beta-D-GalNAc-(1-->4)-alpha-D-Gal-linked positional isomer, were synthesized from 3III,4III-di-O-unprotected globotrioside and the trichloroacetimidate of beta-D-Gal-(1-->3)-beta-D-GalNPhth derivative. Glycosylation at both positions led to the formation of beta-D-GalNPhth-(1-->4)-alpha-D-Gal and beta-D-GalNPhth-(1-->3)-alpha-D-Gal-linked products in a ratio of 1:1 without selectivity. Complete NMR spectral assignments are also described.

Allylmalonamide as a bivalent linker: synthesis of biantennary GM3-saccharide--keyhole limpet hemocyanin glycoconjugate and the immune response in mice.

A biantennary GM3-saccharide (sialyllactoside) derivative (4) was constructed using allylmalonic acid as a bivalent linker, both carboxylic acids of which were condensed with 3-aminopropyl lactoside (2) prior to enzymatic sialylation with a fusion enzyme. While ozonolysis of its allyl group generated a saccharide having a terminal aldehyde (6), we were unable to couple 6 directly to protein by reductive amination. However, extension of the spacer by means of introducing a maleimide group to 6 through its aldehyde group to give 7 enabled the latter to be successfully coupled to thiolated proteins. The average ratios of saccharide to protein were observed to be 35 in KLH conjugate (13) and 9-12 in HSA conjugates (14 and 15). The antisera obtained by immunizing mice with the biantennary sialyllactoside-KLH conjugate (13) together with MPL adjuvant were analyzed by ELISA. Using several structurally related saccharide-HSA conjugates as screening antigens, it was concluded that anti-sialyllactoside antibodies, both IgG and IgM, were effectively raised. This was further supported by competitive inhibition experiments using lactoside (1), sialyllactoside (8) and biantennary sialyllactoside (4) as inhibitors.

Murine immune responses to Neisseria meningitidis group C capsular polysaccharide and a thymus-dependent toxoid conjugate vaccine.

The polysaccharide (PS) capsules of many pathogenic bacteria are poor immunogens in infants and young children as a result of the delayed response to PS antigens during ontogeny. The development of polysaccharide-protein conjugate vaccines for Haemophilus influenzae type b, which have proven to be efficacious in this age group, has led to active development by a number of investigators of conjugate vaccines for other diseases. We describe here the response of several mouse strains to the capsular PS of Neisseria meningitidis group C (MCPS) conjugated to tetanus toxoid (MCPS-TT) and the same response in BALB/c mice as a model of the immune consequences of conjugate vaccine immunization. The use of a conjugate vaccine results in a shift in the isotype elicited in response to the MCPS, from immunoglobulin M (IgM) and IgG3 to primarily IgG1. A response to MCPS-TT is seen even among mouse strains which respond poorly to MCPS itself, emphasizing the importance of a strain survey when choosing a mouse model for a vaccine. The marked increase in IgG1 antibody titer was accompanied by a large increase in bactericidal activity of sera from these animals. Animals primed with the conjugate vaccine demonstrated a booster response after secondary immunization with either the MCPS or the conjugate. The ability to produce a boosted IgG1 anti-MCPS response to the MCPS can be transferred to adoptive recipients by B cells alone from mice primed with MCPS-TT but not mice primed with MCPS alone. These data indicate that in BALB/c mice a single immunization with MCPS-TT is sufficient to induce a shift to IgG1 and generate a memory B-cell population that does not require T cells for boosting.

Crystallization and preliminary X-ray diffraction analysis of antigen-binding fragments which are specific for antigenic conformations of sialic acid homopolymers.

Meningococcal meningitis is a severe childhood disease which often results in significant disability or death. Two major etiological agents of meningitis are the group B meningococci and capsular type K1 E. coli. The virulence of these organisms is attributable to structural mimicry between their common alpha(2-8)-polysialic acid capsular polysaccharide and human tissue antigens, which allows the bacteria to evade immune surveillance. There is currently no effective vaccine to protect against this infection. It has been demonstrated that the capsular polysaccharide of the bacteria can adopt a unique 'antigenic conformation'. This antigenic conformation has formed the basis for the development of an N-propionylated polysialic acid vaccine. Immunization trials in mice with this vaccine show the production of two groups of antibodies, of which only N-propionylated polysialic acid-specific were protective. Knowledge of the structure of the antigen-binding site which recognizes the protective epitope is essential to determining the antigenic conformation of the polysaccharides, and is a critical aspect in understanding and improving the action of potential vaccines. The antigen-binding fragments (Fab) of one protective (13D9) and one non-protective (6B9) monoclonal antibody specific for the capsular polysaccharides of group B meningococci have been crystallized and have undergone preliminary X-ray diffraction analysis. Both crystals are observed to scatter X-rays to approximately 1.7 A resolution at the A1 station at the Cornell High-Energy Synchrotron Source. 13D9 has an orthorhombic unit cell with a = 41.8, b = 102.3, c = 134.7 A, with space group P212121. Fab 6B9 has an orthorhombic unit cell with a = 89.6, b = 132.0 and c = 36.9 A, with space group P21212.

Oligosaccharide fragments of the type III group B streptococcal polysaccharide derived from S. pneumoniae type 14 capsular polysaccharide by a chemoenzymatic method.

Partial N-deacetylation fo the GlcNAc residues in S. pneumoniae type 14 capsular polysaccharide (Pn14-PS) backbone was achieved by treatment with base, and the product was subsequently enzymatically sialylated at the 3-O-positions of the terminal galactose residues. The resultant, partially N-deacetylated type III Group B streptococcus capsular polysaccharide (GBSIII-PS) was subjected to nitrous acid deamination, which resulted in the degradation of GBSIII-PS polysaccharide into oligosaccharides containing increasing numbers of the identical repeating units. The oligosaccharides were then separated by passage through a Superdex 30 column and characterized by ESIMS and NMR spectroscopic analysis.

Streptococcus pneumoniae type 14 polysaccharide-conjugate vaccines: length stabilization of opsonophagocytic conformational polysaccharide epitopes.

A simple and convenient method was developed for the preparation of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.

Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen.

Microbial polysaccharides are notably poor immunogens. We have developed an alternate route for the production of Abs to important carbohydrate epitopes. mAb S9, a protective mAb against the type III capsular polysaccharide of group B streptococci (GBS), was used to select epitope analogues from a peptide display phage library. Depending upon desorption conditions, two populations of phage were identified with displayed sequences of WENWMMGNA and FDTGAFDPDWPA. ELISA results demonstrated that these phage bound to S9 and no other Abs. Phage blocked the binding of S9 to type III GBS, but did not block binding by another anti-GBS mAb. Phage displaying the latter peptide sequence showed greater inhibition. Ab S9 and other monoclonal and polyclonal anti-GBS type III antisera bound the synthetic peptide FDTGAFDPDWPAC. The binding of S9 to GBS was inhibited by the free peptide with an IC50 of 30 microg/ml. The binding of polyclonal anti-GBS antibodies to peptide could be blocked by intact GBS as well as purified capsular polysaccharide. The peptide was conjugated to three different carriers and was used to immunize mice. All mice produced a significant antibody response to GBS and to the purified capsular polysaccharide following a single immunization. These data demonstrate that a peptide mimetic of the GBS capsular polysaccharide is both antigenic and immunogenic. The incorporation of such peptides into vaccine preparations may enhance the efficacy of vaccines in inducing Ab responses to important carbohydrate epitopes.

N-Propionylated group B meningococcal polysaccharide mimics a unique bactericidal capsular epitope in group B Neisseria meningitidis.

The N-propionylated group B meningococcal polysaccharide (NPrGBMP) mimics a unique protective epitope on the surface of group B meningococci (GBM) and Escherichia coli K1. Using a series of monoclonal antibodies (mAbs) induced by the NPrGBMP-monomeric tetanus toxoid (TT) conjugate vaccine it was demonstrated that mAbs having specificities for both extended and conventional short segments of the NPrGBMP were formed, but only the former were bactericidal, and/or gave passive protection against live challenge by GBM. The failure of mAbs specific for short epitopes to protect was further established when (NeuPr)4-TT was used as the vaccine. Of all the mAbs produced that were specific for short internal segments of the NPrGBMP, none were protective, despite the fact that most of them cross-react with the GBM capsular polysaccharide. In contrast, most of the protective mAbs produced by NPrGBMP- TT did not recognize the group B meningococcal polysaccharide (GBMP) unless it was present in its aggregated high molecular weight form. The bactericidal epitope mimicked by the NPrGBMP was shown to be ubiquitous in the capsule of both GBM and E. coli K1 using immunogold labeling techniques and, because of its unique properties, its identification could be significant in the development of a comprehensive conjugate vaccine against group B meningococcal meningitis. This is because most known human alpha(2-8)-polysialic acid self-antigens can be accommodated in 30-50 alpha(2-8)-linked sialic acid residues, which is roughly equivalent to an 11-kD length of the GBMP. It has been hypothesized that the formation of the protective epitope on the surface of GBM is due to the interaction of helical segments of the GBMP with another molecule and that the protective epitope is mimicked by the NPrGBMP. Support for the above hypothesis is provided by the fact that the protective NPrGBMP epitope has a similar unusual length dependency to that of the GBMP epitope.

NMR and molecular dynamics studies of the conformational epitope of the type III group B Streptococcus capsular polysaccharide and derivatives.

The conformational epitope of the type III group B Streptococcus capsular polysaccharide (GBSP III) exhibits unique properties which can be ascribed to the presence of sialic acid in its structure and the requirement for an extended binding site. By means of NMR and molecular dynamics studies on GBSP III and its fragments, the extended epitope of GBSP III was further defined. The influence of sialic acid on the conformational properties of GBSP III was examined by performing conformational analysis on desialylated GBSP III, which is identical to the polysaccharide of Streptococcus pneumoniae type 14, and also on oxidized and reduced GBSP III. Conformational changes were gauged by 1H and 13C chemical shift analysis, NOE, 1D selective TOCSY-NOESY experiments, J(HH) and J(CH) variations, and NOE of OH resonances. Changes in mobility were examined by 13C T1 and T2 measurements. Unrestrained molecular dynamics simulations with explicit water using the AMBER force field and the GLYCAM parameter set were used to assess static and dynamic conformational models, simulate the observable NMR parameters and calculate helical parameters. GBSP III was found to be capable of forming extended helices. Hence, the length dependence of the conformational epitope could be explained by its location on extended helices within the random coil structure of GBSP III. The interaction of sialic acid with the backbone of the PS was also found to be important in defining the conformational epitope of GBSP III.

Structural basis of the Neisseria meningitidis immunotypes including the L4 and L7 immunotypes.

The application of high-resolution 1H, 13C and 31P NMR and MS analyses to the oligosaccharide moieties of the L4 and L7 immunotypes of Neisseria meningitidis revealed that they had the following structures: [formula: see text] The fact that the L7 LPS is not sialylated at O-3 of its terminal beta-D-galactopyranosyl residue implies that it is a mutant strain unable to endogenously sialylate its lacto-N-neotetraose antenna. With the structural elucidation of the L4 and L7 LPS immunotypes, a more comprehensive structural profile of the LPS involved in disease isolates can now be assembled. This provides valuable insights into the structural basis of the N. meningitidis immunotyping system which could be of use in formulating an LPS-based vaccine against meningococcal meningitis.