Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic pentasaccharide fragments using NMR and molecular modeling

As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.     

Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.     

Synthesis of oligosaccharide fragments of O-specific polysaccharides of Shigella flexneri. III. Synthesis of the tetrasaccharide Glc alpha 1-3Rha alpha 1-2(Glc alpha 1-3)Rha alpha 1-OMe and the pentasaccharide GlcNAc beta 1-2(Glc alpha 1-3)Rha alpha 1-2(Glc alpha 1-3)Rha alpha 1-OMe

Two synthetic schemes to prepare the title branched tetrasaccharide and pentasaccharide are described. These oligosaccharides represent fragments of the O-antigenic polysaccharides of Shigella flexneri serotype 5b.     

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.     

O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a

Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of the lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here, we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S. flexneri type 2a LPS. [structure: see text]     

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.     

Development of peptide mimics of a protective epitope of Vibrio cholerae Ogawa O-antigen and investigation of the structural basis of peptide mimicry

As an alternative approach toward the development of a cholera vaccine, the potential of peptide mimics of Vibrio cholerae lipopolysaccharide (LPS) to elicit cross-reactive immune responses against LPS was investigated. Two closely related protective monoclonal antibodies, S-20-4 and A-20-6, which are specific for Ogawa O-antigen (O-specific polysaccharide; O-SP) of V. cholerae O1, were used as the target antibodies (Abs) to pan phage display libraries under different elution conditions. Six phage clones identified from S-20-4 panning showed significant binding to both S-20-4 and A-20-6. Thus, it is likely that these phage-displayed peptides mimic an important conformational epitope of Ogawa antigens and are not simply functionally recognized by S-20-4. Each of the six phage clones that could bind to both monoclonal antibodies also competed with LPS for binding to S-20-4, suggesting that the peptides bind close to the paratope of the Ab. In order to predict how these peptide mimics interact with S-20-4 compared with its carbohydrate counterpart, one peptide mimic, 4P-8, which is one of the highest affinity binders and shares motifs with several other peptide mimics, was selected for further studies using computer modeling methods and site-directed mutagenesis. These studies suggest that 4P-8 is recognized as a hairpin structure that mimics some O-SP interactions with S-20-4 and also makes unique ligand interactions with S-20-4. In addition, 4P-8-KLH was able to elicit anti-LPS Abs in mice, but the immune response was not vibriocidal or protective. However, boosting with 4P-8-KLH after immunizing with LPS prolonged the LPS-reactive IgG and IgM Ab responses as well as vibriocidal titers and provided a much greater degree of protection than priming with LPS alone.     

Structure of the extracellular polysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus 291

The lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus 291, when grown in skimmed milk, produced 80 mg/L exopolysaccharide with an average molecular mass of 1.4 x 10(3) kDa. Monosaccharide analysis, methylation analysis, MS, and 1D/2D NMR (1H and 13C) studies performed on the native polysaccharide, and on oligosaccharides obtained from a mild acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure: [structure: see text].     

Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.     

Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines

The peptides DRPVPY and MDWNMHAA, which were identified as mimics of the cell-surface polysaccharides of Streptococcus Group A and Shigella flexneri Y, respectively, were used in this study to develop experimental vaccines directed against these two bacteria. Both oligopeptides were synthesized employing the Fmoc solid-phase strategy and linked via the amino end to a bifunctional linker, diethylsquarate. These adducts were then conjugated to the two carrier proteins, bovine serum albumin (BSA) and tetanus toxoid (TT) to yield the peptide conjugate vaccines. The average level of incorporation of DRPVPY and MDWNMHAA on TT was 65% and 75%, respectively, whereas that of both peptide haptens on BSA was 100%. A polysaccharide conjugate against S. flexneri Y, which comprises about 10 tetrasaccharide repeating units, was also prepared based on reductive amination at the reducing end with 1,3-diaminopropane, followed by coupling of the aminated polysaccharide to diethylsquarate, and subsequent coupling of the adduct to TT. An average incorporation of 73% of polysaccharide haptens was achieved. The glycoconjugate and the oligopeptide conjugates were shown to bind effectively to the respective monoclonal antibodies directed against the cell-surface polysaccharides.     

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.     

Peptide mimotopes of pneumococcal capsular polysaccharide of 6B serotype: a peptide mimotope can bind to two unrelated antibodies

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of alpha(2-->8) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with alpha(2-8) polysialic acid, but not with 6B PS. Conversely, alpha(2-8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type.     

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.     

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.     

Peptide mimotopes as surrogate antigens of carbohydrates in vaccine discovery

Carbohydrate antigens are immune targets associated with a variety of pathogens and tumor cells. Unfortunately, most carbohydrates are intrinsically T cell-independent antigens, which diminishes their efficacy as immunogens. The conversion of carbohydrate epitopes to peptide mimotopes is one means to overcome the T cell-independent nature of carbohydrate antigens because peptides have an absolute requirement for T cells. Although such conversion has great potential for the development of veterinarian and human vaccines, there are issues related to the use of peptide-based immunogens as functional surrogates. Some of these issues are fundamental, pertaining to how mimicry comes about at the molecular level, and some are application oriented, directed at elucidating important immunological mechanisms. In this article the potential and caveats of this technology regarding its application in vaccine discovery are analyzed.     

Facile synthesis of a pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C

A pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[alpha-D-Galp-(1-->2)-beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->OCH2CH2N3) (1) was synthesized in a regio- and stereoselective manner. The 2-azidoethyl-spacered pentasaccharide mimic 1 can be used to construct a neoglycoconjugate antigen.     

Peptide Libraries Define the Fine Specificity of Anti-polysaccharide Antibodies toCryptococcus neoformans

Cryptococcus neoformansis an encapsulated fungus that causes a life-threatening meningoencephalitis in patients with AIDS. Monoclonal antibodies to the capsular glucuronoxylomannan can modulate the infection in mice, but the epitopes on this complex polysaccharide recognized by protective and non-protective antibodies have not been defined. We have used 2H1, one of our most protective antibodies, to screen phage display peptide libraries for peptide mimotopes that would allow us to explore the fine specificity of anti-cryptococcal polysaccharide antibodies. Hexa- and decapeptides have been identified with sequence homologies that define four motifs: 1, (E)TPXWM/LM/L; 2, W/YXWM/ LYE; 3, DWXDW; and 4, (Ar)WDGQ(Ar). Peptides representing these motifs compete with each other for a shared binding site that overlaps the polysaccharide binding site. Motifs 1 and 2 confer high affinity binding, and PA1, which displays a motif 1 peptide with the sequence LQYTPSWMLV, binds to 2H1 with aK_dof 295 nM. Analysis of the interaction between the 2H1 binding peptides and 24 structurally related anti-polysaccharide antibodies reveals a complex pattern of reactivity that strongly suggests binding to or close to the complementary determining regions. Furthermore, those antibodies that have been shown to have different specificity, and in some cases different protective potential, do not bind any of the peptides selected by the protective 2H1 antibody. This study shows that peptide mimotopes for a complex microbial polysaccharide can be identified by screening phage peptide libraries and demonstrates the usefulness of such peptides in analyzing closely related interactive sites of proteins in general and of antibodies in particular.     

Synthesis of hexa- to tridecasaccharides related to Shigella dysenteriae type 1 for incorporation in experimental vaccines

Hexa- to tridecasaccharides corresponding to the O-specific polysaccharide (O-SP) of the Gram-negative bacterium Shigella dysenteriae type 1 were synthesized in solution phase. The syntheses utilized tetra-, octa-, and dodecasaccharide intermediates that represent one to three contiguous tetrasaccharide repeating units of the O-SP [Synlett2003, 743]. These compounds were glycosylated with mono-, di-, and trisaccharide trichloroacetamidates, which were synthesized in this study. The excellent stereodirecting effect of 4,6-O-benzophenone ketals in glycosylation reactions of 2-azido-2-deoxy-glucopyranosyl donors was demonstrated. The free oligosaccharides were characterized by 1H and 13C NMR spectroscopy and by high-resolution mass spectrometry. The oligosaccharides described herein contain the 5-(methoxycarbonyl)pentyl aglycon for eventual attachment to immunogenic carriers using a recently published protocol [J. Org. Chem.2005, 70, 6987].     

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.     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.