The antigen-binding site of an N-propionylated polysialic acid-specific antibody protective against group B meningococci is consistent with extended epitopes.

Monoclonal antibodies 13D9 and 6B9 are both specific for N-propionylated polysialic acid (NPrPSA); however, while 13D9 is protective against meningitis caused by group B meningococci and Escherichia coli capsular type K1 infection, 6B9 is not. The crystal structures of the Fabs from the two antibodies determined at 2.06 and 2.45 Å resolutions, respectively, reveal markedly different combining sites, where only the surface of 13D9 is consistent with the recognition of extended helical epitopes known to exist in the capsular polysaccharides of etiological agents of meningitis. Interestingly, complementarity determining region H2 on 13D9 lies in a non-canonical conformation that docking studies show is a critical feature in the generation of negative free energy of binding. Finally, the model of extended NPrPSA decasaccharide bound to 13D9 derived from docking studies is consistent with saturation transfer difference nuclear magnetic resonance experiments. Together, these results provide further evidence that extended epitopes have the ability to break immune tolerance associated with the polysialic acid capsule of these pathogens.

Immunization with N-propionyl polysialic acid-KLH conjugate in patients with small cell lung cancer is safe and induces IgM antibodies reactive with SCLC cells and bactericidal against group B meningococci.

PURPOSE: Polysialic acid (polySA) is a polymer side chain bound to the neural cell adhesion molecule that is extensively expressed on the surface of small cell lung cancer (SCLC) cells. In our previous study, a robust antibody response was noted in patients with SCLC after vaccination with 30 µg of keyhole limpet hemocyanin (KLH)-conjugated N-propionylated (NP-) polySA, but peripheral neuropathy and ataxia were detected in several vaccinated patients. The objectives of the current trial were to establish the lowest optimal dose and to confirm the safety of the induction of antibodies against polySA with the NP-polySA vaccine. EXPERIMENTAL DESIGN: Patients with SCLC who completed initial treatment and had no evidence of disease progression were injected with either 10 or 3 µg of NP-polySA conjugated to KLH and mixed with 100 µg of immunologic adjuvant (QS-21) at weeks 1, 2, 3, 4, 8, and 16. RESULTS: Nine patients were enrolled at each of the two dose levels. Prior to vaccination, one patient in each group had low-titer antibodies against polysialic acid. All patients at the 10 µg vaccine dose level responded to vaccination with IgM antibody titers against polysialic acid (median titer 1/1,280 by ELISA), and all but one patient made IgM and IgG antibodies against the artificial vaccine immunogen, NP-polysialic acid (median titer 1/10,240). The antibody responses at the 3 µg vaccine dose level were lower; six of nine patients developed antibodies against polysialic acid (median titer 1/160). Post-vaccination sera from 6/9 and 3/9 patients in the 10 and 3 µg groups reacted strongly with human SCLC cells by fluorescent-activated cell sorting (FACS). Sera from all patients in the 10 µg dose group also had bactericidal activity against group B meningococci with rabbit complement. Self-limited grade 3 ataxia of unclear etiology was seen in 1 of 18 patients. CONCLUSIONS: Vaccination with NP-polySA-KLH resulted in consistent high-titer antibody responses, with the 10 µg dose significantly more immunogenic than the 3 µg dose. This study establishes the lowest optimally immunogenic dose of NP-polysialic acid in this NP-polysialic acid-KLH conjugate vaccine to be at least 10 µg, and it establishes the vaccine's safety. We plan to incorporate NP-polySA into a polyvalent vaccine against SCLC with four glycolipid antigens also widely expressed in SCLC-GD2, GD3, fucosylated GM1, and globo H.

Rational chemical design of the carbohydrate in a glycoconjugate vaccine enhances IgM-to-IgG switching.

Many pathogens are sheltered from host immunity by surface polysaccharides that would be ideal as vaccines except that they are too similar to host antigens to be immunogenic. The production of functional IgG is a desirable response to vaccines; because IgG is the only isotype that crosses the placenta, it is of particular importance in maternal vaccines against neonatal disease due to group B Streptococcus (GBS). Clinical studies found a substantially lower proportion of IgG-relative to IgM-among antibodies elicited by conjugates prepared with purified GBS type V capsular polysaccharide (CPS) than among those evoked by CPSs of other GBS serotypes. The epitope specificity of IgG elicited in humans by a conjugate prepared with type V CPS is for chemically desialylated type V CPS (dV CPS). We studied desialylation as a mechanism for enhancing the ability of type V CPS to induce IgM-to-IgG switching. Desialylation did not affect the structural conformation of type V CPS. Rhesus macaques, whose isotype responses to GBS conjugates match those of humans, produced functionally active IgG in response to a dV CPS-tetanus toxoid conjugate (dV-TT), and 98% of neonatal mice born to dams vaccinated with dV-TT survived lethal challenge with viable GBS. Targeted chemical engineering of a carbohydrate to create a molecule less like host self may be a rational approach for improving other glycoconjugates.

Conjugation of Meningococcal Lipooligosaccharides Through Their Non-Reducing Terminus Results in Improved Induction a Protective Immune Response.

The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-D-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

Vaccination of small cell lung cancer patients with polysialic acid or N-propionylated polysialic acid conjugated to keyhole limpet hemocyanin.

PURPOSE: Long chain polysialic acid (polySA) is a side chain on embryonal neural cell adhesion molecules that, in the adult, is largely restricted to small cell lung cancer (SCLC). Long chains of polySA are also expressed on group B meningococcus. In this clinical trial, we aimed to elicit an immune response against polysialic acid to target clinically inapparent residual disease in patients with SCLC who had successfully completed initial therapy. EXPERIMENTAL DESIGN: Patients were vaccinated with either 30 micro g unmodified polySA or N-propionylated-polySA (NP-polySA), conjugated to keyhole limpet hemocyanin (KLH) and mixed with 100 micro g of immunological adjuvant QS-21 at weeks 1, 2, 3, 4, 8, and 16. RESULTS: Of the 5 evaluable patients vaccinated with unmodified polySA, only 1 mounted an IgM antibody response to polySA. On the other hand, all 6 of the patients vaccinated with NP-polySA produced IgM antibodies to NP-polySA and these cross-reacted with unmodified polySA in all but 1 case. IgG antibodies to NP-polySA were observed in 5 of the patients, but these did not cross-react with polySA. The presence of IgM antibodies reactive with SCLC cell lines was confirmed in this group by flow cytometry. Complement-dependent lysis of tumor cells could not be demonstrated. However, postimmunization sera induced significant bactericidal activity against group B meningococcus when combined with rabbit complement. CONCLUSIONS: Vaccination with NP-polySA-KLH, but not polySA-KLH, resulted in a consistent high titer antibody response. We are now conducting a de-escalation dosing study with NP-polySA-KLH to better assess the immunogenicity, toxicities, and optimal dose of this vaccine. We plan to incorporate this vaccine as a component of a polyvalent vaccine with GM2, fucosylated GM1, and Globo H to target SCLC.

Characterization of polysaccharide conformational epitopes by surface plasmon resonance.

SPR techniques can provide a wealth of insight into the nature of protein-carbohydrate interactions. Information not obtained readily by other methodologies can be gathered relatively quickly in a label-free manner with low sample consumption. This chapter focused on two applications in which SPR has been used to map conformational epitopes on bacterial polysaccharides recognized by protective antibodies. In one example, methods for demonstrating the conformational nature of the epitope recognized by an anti-GBS antibody were described. Dramatic epitopic stabilization at 2 repeating units with further significant stabilization between 7 and 20 repeating units was demonstrated. In a second example, SPR methods were employed in characterization of the epitope recognized by a protective antibody against the group B meningococcus. It was shown that the antibody bound a long epitope, in excess of eight monosaccharides and probably helical, on NPr-GBMP but did not bind to GBMP. The binding of the protective antibody to GBMP only when GBMP is cell associated, or with an attached lipid, indicated that the protective GBM epitope consists of more than GBMP. NPr-GBMP mimics a cell surface complex consisting of extended helical portions of the GBMP in association with a second molecule, possibly a phosphoglycerolipid. SPR experiments indicated that the protective nature of certain antibodies induced by the NPr-GBMP vaccine is attributable to their recognition of an abundant internal epitope on NPr-GBMP and cell-associated GBMP. A nonprotective antibody, specific for NPr-GBMP, recognized a terminal and consequently minor epitope on the polysaccharide. Low levels of this nonprotective antibody binding to cells and unpurified polysaccharide confirmed recognition of a minor epitope on the natural antigen as well. In contrast, a protective antibody exhibited a high level of binding to the cell-associated antigen.

Conjugation of meningococcal lipooligosaccharides through their lipid A terminus conserves their inner epitopes and results in conjugate vaccines having improved immunological properties.

The importance of conserved inner saccharide epitopes to the immune performance of meningococcal lipooligosaccharide-protein conjugate vaccines was demonstrated in the following experiments. Two different oligosaccharides were obtained by chemical degradations of the same L7 lipooligosaccharide, and both were linked terminally to tetanus toxoid. One was a truncated oligosaccharide in which the inner epitopes were incomplete and was obtained by mild acid hydrolysis of the L7 lipooligosaccharide. This oligosaccharide was conjugated by direct reductive amination through its newly exposed terminal Kdo residue. The second, a full-length oligosaccharide, was obtained by O-deacylation of the L7 lipooligosaccharide, with subsequent removal of phosphate substituents from its lipid A moiety using alkaline phosphatase. This permitted the full-length oligosaccharide to be conjugated directly to tetanus toxoid by reductive amination through its newly exposed terminal 2-N-acyl-2-deoxy-D-glucopyranose residue. Comparison of the immune performance of the two conjugates in mice revealed, that while both were able to induce significant levels of L7-lipooligosaccharide-specific IgG antibody, the conjugate made with the full-length saccharide was able to induce antibodies with increased bactericidal activity against homologous meningococci.

Sialyltransferase inhibitors: consideration of molecular shape and charge/hydrophobic interactions.

In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltransferase. The first type of compounds were designed as substrate mimics in which the phosphate in CMP-Neu5NAc was replaced by a non-hydrolysable, uncharged 1,2,3-triazole moiety. The second type of compound contained a 2-deoxy-2,3-dehydro-acetylneuraminic moiety which was linked to cytidine through its carboxylic acid and amide linkers. In the third type of compound the sialyl phosphate was substituted by an aryl sulfonamide which was then linked to cytidine. Inhibition study of these cytidine conjugates against Campylobacter jejuni sialyltransferase Cst 06 showed that the first type of molecules are competitive inhibitors, whereas the other two could only inhibit the enzyme non-competitively. The results indicate that although the binding specificity may be guided by molecular shape and H-bond interaction, the charge and hydrophobic interactions contributed most to the binding affinity.

Fine specificity and cross-reactions of monoclonal antibodies to group B streptococcal capsular polysaccharide type III.

Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant "protective" immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPS(III) mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPS(III) mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen.

Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment.

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.

Understanding the bacterial polysaccharide antigenicity of Streptococcus agalactiae versus Streptococcus pneumoniae.

Bacterial surface capsular polysaccharides (CPS) that are similar in carbohydrate sequence may differ markedly in immunogenicity and antigenicity. The structural origin of these phenomena is poorly understood. Such a case is presented by the Gram-positive bacteria Streptococcus agalactiae (Group B Streptococcus; GBS) type III (GBSIII) and Streptococcus pneumoniae (Pn) type 14 (Pn14), which share closely related CPS sequences. Nevertheless, antibodies (Abs) against GBSIII rarely cross-react with the CPS from Pn14. To establish the origin for the variation in CPS antigenicity, models for the immune complexes of CPS fragments from GBSIII and Pn14, with the variable fragment (Fv) of a GBS-specific mAb (mAb 1B1), are presented. The complexes are generated through a combination of comparative Ab modeling and automated ligand docking, followed by explicitly solvated 10-ns molecular dynamics simulations. The relationship between carbohydrate sequence and antigenicity is further quantified through the computation of interaction energies using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method, augmented by conformational entropy estimates. Despite the electrostatic differences between Pn14 and GBSIII CPS, analysis indicates that entropic penalties are primarily responsible for the loss of affinity of the highly flexible Pn14 CPS for mAb 1B1. The similarity of the solution conformation of the relatively rigid GBSIII CPS with that in the immune complex characterizes the previously undescribed 3D structure of the conformational epitope. The analysis provides a comprehensive interpretation for a large body of biochemical and immunological data related to Ab recognition of bacterial polysaccharides and should be applicable to other Ab-carbohydrate interactions.

Novel zinc (II)-mediated epimerization of 2'-carbonylalkyl-alpha-C-glycopyranosides to their beta-anomers.

2'-Aldehydes and 2'-ketones of alpha-C-glycosides, including the gluco-, galacto-, and manno- series, were epimerized exclusively to their beta-anomers in good-to-excellent yields under basic conditions and in the presence of zinc acetate. The beta-stereoselectivity is independent of the neighboring group at 2-O-substitution of sugar substrates. Therefore, this provides a particularly useful method for the preparation of manno-beta-C-glycosides. The epimerization is likely initiated by the formation of Zn-enolate that is stabilized by intramolecular chelation to the pyranose ring-oxygen to form a syn chair-boat structure. Due to the activation generated by the Zn-O coordination, fission of the C1-O bond occurs, leading to opening of the pyranose ring, which is spontaneously followed by a change in conformation. The more stable anti chair-boat transition state is favored, and the subsequent hetero-intramolecular Michael addition results in the formation of beta-C-glycoside in a ring-closure step.

Epimerization of 2'-carbonylalkyl-C-glycosides via enolation, beta-elimination and intramolecular cycloaddition.

Treatment of 2'-carbonyl-alpha-C-glycopyranosides of gluco, galacto, manno, 2-deoxy, and 2-azido sugars with 4% NaOMe resulted in anomeric epimerization to give their respective beta-anomers in good to excellent yields. The epimerization of the 2'-aldehyde of alpha-C-galactopyranoside (10) in deuterium methanol, which afforded the beta-anomer with exclusive deuterium replacements at the 1'-position, excluded the possibility of the exo-glycal as being involved as an intermediate. When 2'-aldehyde (36) and 2'-ketone (41) of 2,3-di-O-benzyl-alpha/beta-l-C-arabinofuranoside were used as substrates we were able to obtain the respective equatorial alpha-C-arabinopyranosides (37 and 42). These observations confirmed that the epimerization involves an acyclic alpha,beta-unsaturated aldehyde or ketone, which is formed by the enolation of 2'-carbonyl-alpha-C-glycoside with subsequent beta-elimination. Thereafter an intramolecular hetero-Michael cycloaddition occurs, leading to the formation of thermodynamically controlled stable products, which were exclusively the equatorial C-glycopyranosides, except in the case of 2'-carbonyl-C-furanosides, where a mixture of two anomers was obtained.

Discovery of an alpha 2,9-PolyNeu5Ac glycoprotein in C-1300 murine neuroblastoma (clone NB41A3).

alpha2,8-PolyNeu5Ac is expressed on neural cell adhesion molecules during embryogenesis and also re-expressed on certain tumors. PolyNeu5Ac is therefore an oncodevelopmental antigen, has important regulatory effects on the adhesive and migratory behavior of neural cells, and is thus crucial to synaptic plasticity. Until now, alpha2,9-polyNeu5Ac, a linkage isomer of alpha2,8-polyNeu5Ac, has long been thought to occur only in capsules of neuroinvasive Neisseria meningitidis group C bacteria. Here we report the unexpected discovery of alpha2,9-polyNeu5Ac in a new cell adhesion-related glycoprotein on the membrane of C-1300 murine neuroblastoma cells (clone NB41A3). We also report the expression of alpha2,9-polyNeu5Ac was affected by cell growth and retinoic acid-induced differentiation. Occurrence of the linkage isomer of alpha2,8-polyNeu5Ac has been left unrecognized by conventional methods using biological diagnostic probes for alpha2,8-polyNeu5Ac. Thus, our discovery may change contemporary views of biology and pathology of polysialic acid and open new avenues for the development of anti-neural tumor drugs.

Bioengineering of surface GD3 ganglioside for immunotargeting human melanoma cells.

N-Propionyl, N-butyryl (N-Bu), and N-benzoyl mannosamine, as precursors of sialic acid biosynthesis, were incubated with human melanoma SK-MEL-28 cells and resulted in the replacement of N-acetyl groups on the cell surface sialic acid residues, including those associated with GD3. Meanwhile, vaccines containing GD3 and modified GD3 tetrasaccharide-keyhole limpet hemocyanin conjugates were synthesized, and BALB/c mice were immunized with them together with monophosphoryl lipid A adjuvant. The GD3Bu-keyhole limpet hemocyanin conjugate raised the highest IgG titers without any cross-reactivity to unmodified GD3. Expression of GD3Bu epitopes on the surface of SK-MEL-28 cells was confirmed in vitro and in vivo by the binding of a polyclonal antiserum and monoclonal antibody (mAb) 2A, both of which specifically recognize GD3Bu, and by mass spectroscopic analysis of glycolipids extracted from cells. Following expression of GD3Bu on the surface of SK-MEL-28 cells, the cells could be lysed by mAb 2A and GD3Bu antiserum in the presence of complement. Although less effective in the control of existing large size tumors ( approximately 10 mm inner diameter) on BALB/c nu/nu mice, mAb 2A in combination with ManNBu effectively protected mice from SK-MEL-28 tumor grafting. This approach may provide a method to augment the immunogenicity of sialylated human antigens and to avoid generating an autoimmune response to them at same time.

Induction of cross-reactive antibodies by immunization of healthy adults with types Ia and Ib group B streptococcal polysaccharide-tetanus toxoid conjugate vaccines.

Types Ia and Ib group B streptococcal (GBS) capsular polysaccharides (PSs) are structural isomers but are antigenically distinct. Immunization of healthy adults with GBS type Ia PS-tetanus toxoid (Ia-TT) or Ib-TT glycoconjugate vaccines induced > or = 4-fold increases in specific immunoglobulin G to the heterologous PS in more than two-thirds of subjects. Ib-TT vaccine-induced IgG bound with substantially higher affinity to homologous (Ib) than to heterologous (Ia) PS and promoted opsonophagocytic killing of GBS type Ib but not type Ia organisms. The failure of the Ib-TT- and Ia-TT-induced human antibodies to kill bacteria of the cross-reactive serotype contrasts with the results of previous studies in animals. Inhibition enzyme-linked immunosorbent assays demonstrated that Ib-TT-induced IgG to the homologous PS bound mainly to native Ib PS, whereas the cross-reactive antibodies recognized both native and derivative PSs. These results indicate that GBS Ia and Ib PSs should be included in a multivalent conjugate vaccine to prevent GBS disease.

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody.

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.