Rachel Schneerson, MD, Head, Section on Bacterial Disease Pathogenesis and Immunity
John B. Robbins, MD, Principal Investigator
Chiayung Chu, MD, Staff Scientist
Vince Pozsgay, PhD, Staff Scientist
Shousun C. Szu, PhD, Staff Scientist
Bruce Coxon, PhD, Senior Research Fellow
Victor Nelson, PhD, Senior Research Fellow
Peter Ftacek, PhD, Visiting Fellow
Agnieszka Korzeniowska-Kowal, PhD, Visiting Fellow
Zhigang Jin, MD, Research Fellow
Joanna Kubler-Kielb, PhD, Research Fellow
Gil Ben-Menachem, PhD, Postdoctoral Fellow
Patrick Claude, PhD, Postdoctoral Fellow
Carl G. Ekborg, PhD, ORISE ¹ Fellow
Kimi F. Lin, PhD, ORISE ¹ Fellow
Darrell T. Liu, PhD, ORISE1 Fellow
Chunyan Guo, MD, Biologist
Fathy D. Majadly, ASc, BS, Senior Research Assistant
Steven W. Hunt, BSc, Technician
Christopher P. Mocca, BS, Technician
Loc B. Trinh, BSc, Technician
Elizabeth Ogbonna, BSc, BA, Charles River Research Assistant
Arthur B. Karpas, PhD, Guest Researcher
Jianping Li, MD, Guest Researcher

Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs), serve as both essential virulence factors and protective antigens. The age-related and T cell-independent immunogenicity of CPSs limit their use as vaccines, especially in infants and young children. LPSs are too toxic to be administered. Accordingly, the O-specific polysaccharides (O-SPs) of LPSs, which share the virulence-promoting and protectiveness properties of CPSs, must be purified. However, O-SPs are too small to be immunogenic (haptens). Covalent binding of CPSs or O-SPs to medically useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence on these saccharides. Part of the work of our laboratory focuses on making synthetic vaccines that improve immunogenicity.

Polysaccharide/oligosaccharide-protein conjugates

Schneerson, Robbins, Ben-Menachem, Chu, Coxon, Guo, Jin, Kubler-Kielb, Majadly, Mocca, Pozsgay, Trinh, Claude, Ekborg, Karpas, Liu, Nelson, Ogbonna; in collaboration with Ashkenazi, Bohach, Claesson, Freedberg, Glatman-Freedman, Gottfredsson, Grove Krause, Hoogerhout, Lagergard, Leppla, Miller L, Miller M, Nguyen, Passwell, Shiloach, Vinogradov

Shigellae.

We found that the O-SP of Shigella sonnei, bound to recombinant non-toxic P. aeruginosa exoprotein A (rEPA), and the O-SP of S. flexneri 2a, bound to the succinylated, exoprotein A (rEPA-succ), were safe and induced IgG antibodies to the homologous LPS in one- to four-year-olds. A randomized, blinded, Phase 3 study of these conjugates in one- to four-year-olds, with each conjugate serving as a control for the other, is in progress. We have thus far studied the effect of the children's immune sera and IgG isolated from them on Shigella invasion into epithelial intestinal cells in vitro by using Caco-2 and HeLa cells. The sera inhibited Shigella invasion in a type-specific manner. Pretreatment of the sera or Caco-2 cells with O-SP abrogated these effects in a type-specific and dose-dependent manner.

Modification of O-SP by glycosylation or acetylation is important for its antigenic specificity and immunogenicity. We investigated the degree of O-acetylation and localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP. The structure of the repeat unit was assigned.

Cross-reacting polysaccharides (H. influenzae type b [Hib] and type a [Hia], Bacillus pumilus).

Sharing a D-1,5-ribitol phosphate in its repeat unit with Hib, Hia was the (distant) second most common H. influenzae isolate from patients, causing up to 10 percent of disease in some populations. Its importance has risen with the successful control of Hib disease. To provide a potential vaccine for the prevention of Hia disease, we isolated its CPS and conjugated it to a recombinant S. aureus enterotoxin C1 (rSEC). Injected subcutaneously as a saline solution into five- to six-week-old mice, the conjugate induced CPS antibodies with bactericidal activity.

The cell wall polysaccharide (PS) of B. pumilus Sh 18 was reported to cross-react with Hib CPS, presumably as a consequence of its D-ribitol phosphate, which is a common component of bacilli and staphylococcal cell wall PS. Anti-Hib CPS was also shown to precipitate with the cell wall PS of L. plantarium and S. aureus. We isolated the cell wall PS of B. pumilus Sh18 and investigated its structure by using gas chromatography-mass spectrometry, nuclear magnetic resonance (NMR), fast atom bombardment, and several sugar-degrading techniques. We showed the cell wall to be composed of polyribitolphosphate (poly Rib-P), poly Rib-P substituted at C2 with N-acetyl glucosamine, and polyglycerolphosphate (Gly-P); however, we failed to separate these components. The preparation bound not only to anti-Hib but also to anti-S. epidermidis, which is known to contain poly (gly-P) in its teichoic acid. We conjugated the Sh18 PS to carrier proteins and evaluated its immunogenicity in general-purpose mice. Conjugate-induced antibodies reacted with the homologous and several cross-reacting polysaccharides. We synthesized poly Rib-P and conjugated an octamer and a dodecamer with terminal keto groups to aminooxylated bovine serum albumin (BSA) or tetanus toxoid. MALDI-TOF showed that 10 to 18 saccharide chains were incorporated per mole of BSA. We are currently studying the immunogenicity of these conjugates.

Group B Neisseria meningitidis and Escherichia coli K1.

Group B meningococcus (GBM) and Escherichia coli K1 meningitis continue to cause serious diseases that are difficult to treat, and there is no licensed vaccine for their prevention. The polysaccharide alpha(2-8)N-acetylneuraminic acid (PSA) is the CPS of GBM and of E. coli K1 and a component of mammalian glycopeptides. Given that PSA is a "self antigen," vaccines designed to induce PSA immune responses have been considered a potential cause of immunopathology. Antibodies to PSA bind to human tissues in vitro but have not been shown to induce pathology in vivo. The incidence, severity, and nature of systemic infection caused by GBM are similar to those caused by the closely related group C meningococci (GCM). As shown for other polysaccharides, humans demonstrate an age-related acquisition of serum PSA antibodies: most human neonates and adults have IgG anti-PSA. Purified PSA is not immunogenic, but, as a component of bacteria or bound to proteins as a conjugate, this CPS induces antibodies of the three major isotypes, with its IgM and IgG components protective in in vitro and in vivo models. Despite PSA's similarity to mammalian glycoproteins, no data indicate an association of IgG anti-PSA with immunopathology in experimental animals or in humans. A follow-up of about 50,000 person years of individuals with a history of meningococcal infection in Denmark and Iceland found no increased association with autoimmune disease in patients with GBM disease as compared to those with group C meningococcal disease. We propose consideration of clinical trials of PSA conjugate vaccines shown to be immunogenic and protective in animals.

Borrelia burgdorferi.

B. burgdorferi, a spirochete transmitted through the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against it is not effective under age 12 and therefore was recently taken off the market. LPS has been described in other spirochetes, but its presence in B. burgdorferi has been debated. We have not been able to confirm its presence, although our search for LPS revealed two unique glycolipids: cholesteryl 6-O-acyl-beta-D-galactopyranoside (BbGL I) and 1,2-di-O-acyl-3-O-alpha-D-galactopyranosyl-sn-glycerol (BbGL II). Evidence suggests that both are surface-exposed. Injected in various formulations into mice, BbGL I induced specific antibodies (in order of induced levels: CFA, PBS, DMSO, and squalene). Investigation of the effect of BbGL-I and -II on human polymorphonuclear leukocytess and monocytes showed increased levels of IFN-gamma, IL-4, and TNF-alpha secreted in the glycolipids' presence. The cholesteryl palmitoyl galactopyranoside was synthesized with an aldehydo group at the palmitoyl end, which was used to bind to aminooxylated BSA. An average 18 glycolipid groups were incorporated per BSA molecule. The immunogenicity of these conjugates is under study in mice.

Bordetellae.

Pertussis, which is whooping cough in infants, is caused by B. pertussis. B. parapertussis causes pertussis in humans, though of a milder form and lower frequency than B. pertussis. B. bronchiseptica causes respiratory infection in animals, rarely in humans. B. parapertussis and B. bronchiseptica do not produce pertussis toxin. The Lipid A and core regions of the three Bordetella species are similar, but only B. parapertussis and B. bronchiseptica possess an O-SP, the structure of which was reported; however, linkages to the core PS were not identified. We analyzed the carbohydrate structures of the LPS from several strains of Bordetella bronchiseptica and Bordetella parapertussis, using different chemical degradation methods, NMR spectroscopy, and mass spectrometry, and identified a novel pentasaccharide that links the O-chain to the core in all LPSs studied. In addition, whereas the O-chain of these bacteria was reported to be composed of a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, we discovered that it contains amidated uronic acids, the number of which varies among strains.

Haemophilus ducreyi.

H. ducreyi is the cause of chancroid, a sexually transmitted disease characterized by painful genital ulceration. Chancroid also enhances the spread of HIV infection. A major virulence factor and a potentially protective antigen of H. ducreyi is its lipo-oligosaccharide (LOS). To prepare an experimental LOS-based vaccine, we isolated the LOS, hydrolyzed its lipid A, and bound the O-SP to carrier proteins by two new methods: (1) binding the carbonyl group at the Kdo (3-deoxy-d-manno-2-octulosonic acid) of the reducing end of the O-SP to an aminooxy group of a bifunctional linker, which in turn was bound to carrier proteins; and (2) using adipic acid dihydrazide to bind to the carbonyl group of the O-SP and to benzaldehyde-derivatized carrier proteins. In both cases, the functional group was on the reducing end of oligosaccharide (OS), preserving the external non-reducing end structure of the OS. Given that the LOS of H. ducreyi consists of the core region only, acid hydrolysis may potentially modify the native epitope, as was reported for Chlamydia LOS. Thus, we prepared additional conjugates of hydrazine-treated LOS and will evaluate their immunogenicity in mice.

Peptide-protein conjugate vaccines

Schneerson, Robbins, Liu, Majadly, Mocca, Guo; in collaboration with Bellanti, Casadevall, Leppla, Miller L, Nussenzweig, Singer

Bacillus anthracis.

B. anthracis, a potential cause of lethal human infection, has two essential virulence factors without either of which it is not pathogenic for humans: the anthrax toxin and the capsule. The toxin is composed of three peptides: Lethal Factor (LF), Edema Factor (EF), and Protective Antigen (PA). PA is the toxin part that binds to mammalian cells. A 20 KDa peptide must be hydrolyzed off PA, exposing a site to which LF or EF may bind, rendering toxins that enzymatically modify substrates in the mammalian cell cytosol. The capsule is composed of poly-D-gamma-glutamic acid (PGA). It is non-immunogenic and its protective is effect not clear. The licensed PA-based vaccine is safe and protective but has limitations that justify development of improved vaccines. We isolated a recombinant PA from an uncapsulated strain and found that several formulations with formaldehyde-treated and alum-adsorbed materials are immunogenic in mice. Clinical evaluation of these formulations is under way. We isolated the capsule from a non-toxic strain and bound it or corresponding synthetic peptides to BSA, rEPA, rPA, or tetanus toxoid. Thioether, hydrazone, and oxime linkages between the gamma D-PGA and the proteins, with active groups at the C- or N-termini, yielded conjugates that were immunogenic in mice, with no statistical difference between them. The antibodies were opsonophagocytic. Peptides 10-20-mers long and 10-15 mole gamma D-PGA per mole protein were the most immunogenic. The use of alum adjuvant increased PA antibody levels with little effect on anti-PGA levels. Serum IgG anti-PA was measured in 246 sera of recruits injected with the Anthrax Vaccine Adsorbed (AVA) stored at the Department of Defense Serum Repository. Serum conversion rates of four-fold increase in antibody levels were as follows: pre-/post-third 85.3 percent; pre-/post-fourth 67.9 percent; and pre-/post-sixth 45 percent. Geometric mean levels of all individuals were 59.9 microgr/mL following the third injection, 157.4 μg/mL following the fourth, and 277 μg/mL following the sixth.

Plasmodium falciparum.

Malaria is a leading cause of global morbidity and mortality, especially in children; it is estimated to cause over 1 million childhood deaths annually. P. falciparum causes the most severe form of the disease. Experimental vaccines have been described and some tested clinically, but no licensed vaccine is available. The most studied vaccine is the circumsporozoite protein (CS), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were found to be safe and immunogenic but poorly protective, even when administered with adjuvants. Drawing on our studies with peptides of the B. anthracis capsule, we synthesized peptides of four repeat units of NANP bound to carrier proteins and studied their immunogenicity in general-purpose mice without adjuvants and by a scheme suitable for humans. The conjugates induced high levels of antibodies, with circumsporozoite-neutralizing activity roughly correlated with levels measured by ELISA. In another approach designed to provide a transmission-blocking vaccine, we used several methods (amide, hydrazone, or thioether linkages) to bind Pfs25—a low molecular-weight protein, non-immunogenic by itself—to itself or carrier proteins and injected it into mice to evaluate their antibody responses. All conjugates were immunogenic with booster responses upon reinjection. The most effective immunogens were created by using adipic acid dihydrazide as the linker.

Protein and polysaccharide conjugate vaccines to enteric diseases

Szu, Li, Lin, Ftacek, Korzeniowska-Kowal, Hunt; in collaboration with Ahmed, Clements, Hanson, Hoshino, Cao, Kapikian, Kopecko, Moore, Prentice, Robertson, Shiloach

Surface polysaccharides of Gram-negative enteric pathogens, capsules, or lipopolysaccharides are both essential virulence factors and protective antigens. Their immunogenicity is enhanced by binding them to carrier proteins. Viral surface capsid proteins are commonly found to be protective antigens and suitable as vaccine candidate.

Salmonella typhi.

Vi, the capsular polysaccharide of Salmonella typhi, is an essential virulence factor and a protective antigen. To improve its immunogenicity in young children, we conjugated Vi to a carrier protein, the recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A Phase 3 trial of the Vi-rEPA conjugate vaccine in about 12,000 two- to five-year-old children showed an efficacy of 89 percent after 48 months of surveillance. A clinical trial of Vi-rEPA injected together with DTP in Vietnamese infants has been approved; the trial began in March 2006, with 50 infants enrolled since August 2006. We also studied the effect of low birth weight on the immune response in adulthood, using the Vi polysaccharide from S. typhi as a probe. Substantial evidence links low birth weight (not due to prematurity) to susceptibility to chronic disease later in life. Working from the premise that components of the immune system may be programmed early in life, we investigated the potential association between birth weight and response to vaccination with Vi in a cohort of 257 adults (mean age: 29.4 years; 146 men) born in an urban slum in Lahore, Pakistan, during 1964-1978. Primary immunization with Vi showed a correlation between birth weight and IgG anti-Vi response, with lower birth weight associated with lower antibody levels. These adults were re-injected with the same vaccine two years later, with no change in results. In contrast, we observed no birth weight/antibody response relation in a comparable group of adults injected with T cell-dependent antigens such as Hib-conjugate and rabies vaccines.

Salmonella paratyphi A.

S. paratyphi A is the second most common cause of enteric fever in Southeast Asia. A conjugate vaccine of the O-SP of S. paratyphi A and tetanus toxoid was found to be safe and immunogenic in adults, teenagers, and two- to four-year-old children. We found that the O-acetyl content of the PS was directly related to the immunogenicity of the conjugate. To enhance the immunogenicity of the O-SP by increasing its molecular weight, we constructed a mutant S. paratyphi A by replacing the LPS elongation chain-length regulator, the wzz gene, with that of E. coli K12. Silver staining of an SDS-PAGE of this O-SP showed a higher molecular weight distribution than that of the native LPS.

Escherichia coli O157.

E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. A Phase 1 trial of its O-SP-rEPA conjugate demonstrated safety and immunogenicity in adult volunteers. In a Phase 2 study of the conjugate, 55 two- to five-year-old children were injected once or twice. The vaccine was found to be safe and immunogenic; 98 percent of children responded with a greater than four-fold rise in IgG anti-LPS by six months after the first injection, with no booster response after a second injection.

Enterotoxigenic Escherichia coli (ETEC).

E. coli that secrete cholera-like toxin (ETEC) cause diarrhea in most countries, although the incidence, morbidity, and mortality of ETEC diarrhea are highest in developing countries. ETEC diarrhea is caused by two exotoxins: the first, similar in structure and action to the cholera toxin, is known as the heat-labile toxin (LT); and the second is a polypeptide of 19 amino acids called heat-stable toxin (ST). About 40 percent of ETEC in strains from patients secrete LT alone, 20 percent secrete both LT and ST, and 60 percent secrete ST alone. The immunogenicity or protective actions of LT and ST have not been studied thus far. We obtained purified LT and a recombinant mutant non-toxic protein (rLT) from John Clements. The purified LT and rLT, treated with low concentrations of formaldehyde, were injected into young outbred mice. Both induced high levels of IgG anti-LT. We observed a dose-dependent swelling at the injection site. Adsorption of the proteins onto aluminum hydroxide delayed this adverse reaction.

Vibrio cholera O1 and O139.

Vibrio cholera O1 and O139 are the major disease-causing serotypes. The CPS of V. cholera O139 or the O-SP of V. cholera O1 conjugated to various carrier proteins elicited vibriocidal antibodies in mice, at higher levels than those induced by the LPS alone.

Campylobacter jejuni.

Campylobacter jejuni infection is one of the most common enteric infections around the world. Most infections are mild, but serious complications, notably Guillain-Barré syndrome, may occur. Campylobacter is microaerophilic; thus, fermentation in liquid media has been difficult. To design an experimental vaccine based on its LOS, we investigated culture conditions, such as various media and fermentation, and established requirements for growth in liquid media. We then cultivated several C. jejuni serotypes. Chemical analysis of the LOS showed several keto groups. Protein conjugates of the LOS or of the deacylated LOS were immunogenic in mice, and the antibodies elicited by these conjugates showed complement-dependent bactericidal activity. We analyzed 28 isolates of C. jejuni from pediatric patients in Israel for serotype distribution based on the HS (capsule) scheme and found a diverse serotype distribution. Chemical and serological analysis of the LOS of these isolates revealed no correlation between HS types and LOS carbohydrate compositions. Furthermore, approximately 40 percent of the 16 chemically analyzed isolates contained sialic acid in their LOS and may thus bind to antisera against gangaliosides.

Rotavirus.

Rotavius infection is the most common cause of infantile diarrhea regardless of economic status. Vaccine development strategies have mostly followed the approach of the reassortant whole-cell oral vaccine. One such vaccine, Rotashield, was withdrawn after a suspected increase in the incidence of intussusception following vaccination. We have initiated a study of the surface proteins of capsids VP8 and VP7 as immunogens.

Synthetic vaccines, based on synthetic glycoconjugates, against human pathogenic bacteria

Pozsgay, Schneerson, Robbins, Ekborg, Nelson, Claude, Kubler-Kielb

Our studies aim at designing and developing bacterial vaccines. Human bacterial pathogens may have surface-exposed saccharides that serve as both virulence factors and protective antigens. Such saccharides include CPSs, LPSs, and cell wall polysaccharides, which may all vary in size and complexity. We are studying synthetic chemical approaches to bacterial surface-exposed saccharide structures that can be used to elicit serum IgG antibodies against bacterial pathogens.

Synthetic vaccine against Shigella dysenteriae type 1.

Shigella dysenteriae type 1, a Gram-negative human pathogen, causes dysentery and diarrhea in endemic and epidemic forms in developing countries. The lack of a licensed vaccine against this organism and the emergence of strains increasingly resistant to available antibiotics call for the development of a vaccine. Our approach is based on immunogens that induce serum IgG antibodies to the O-SP of the LPS. Such an immunogen was shown to be protective against S. sonnei dysentery in adults. The O-SP of S. dysenteriae type 1 consists of approximately 100 monosaccharide residues. The repeating unit of the O-SP is a tetrasaccharide that contains two L-rhamnose residues and one N-acetyl-D-glucosamine and one D-galactose residue. We have demonstrated that an oligosaccharide as small as an octasaccharide made up of two contiguous Rha-Gal-GlcNAc-Rha sequences can elicit O-SP-specific IgG antibodies in mice when covalently linked to an immunogenic protein. The highest immune responses were obtained with conjugates containing an average of 10 to 12 copies of dodeca- and hexadecasaccharides, with the above sequence, per human serum albumin used as a carrier protein. We are also studying the importance of the non-reducing terminal monosaccharide residue in eliciting an O-SP immune response in mice. To that end, we synthesized spacer-linked hexa- to pentadecasaccharides corresponding to the sequence above, exposing each of the component monosaccharides at the non-reducing end of the saccharide constructs. All the synthetic oligosaccharides inhibited 20 to 25 percent binding of the LPS to polyclonal IgG mouse anti-O-SP; that is, neither the saccharide chain length nor identity of the terminal monosaccharide residue had a significant role in inhibition. However, as immunogens, conjugates of the oligosaccharides with bovine serum albumin elicited O-SP serum antibodies in mice in a structure-dependent fashion; the constructs with either N-acetyl-D-glucosamine or D-galactose at their non-reducing end elicited approximately 10 times higher IgG levels than those terminating in either of the two rhamnose residues. To explore further the effect of the terminal monosaccharide on O-SP immunogenicity, we synthesized oligosaccharides of equal length but different terminal sugars and conjugated them to the recombinant exotoxin A of Pseudomonas aeruginosa.

Synthetic vaccine against Borrelia burgdorferi.

B. burgdorferi, the etiological agent of Lyme disease, expresses on its surface a unique glycolipid that is composed of palmitoyl-galactosyl-cholesterol. Given its surface location on the bacterium, this unusual glycolipid may be a candidate for vaccine development. Its chemical synthesis has been reported. Moreover, we have studied the glycolipid's chemical synthesis in a form that allows its covalent attachment to proteins. We used the previously reported galactosyl cholesterol that was chemically modified so as to allow the attachment of a palmitic acid moiety at position O-6 of the galactose unit. This moiety also features a latent aldehydo group at its end. After removal of all the protecting groups, the aldehydo-modified glycolipid was covalently attached to amino-oxypropylated BSA, using the reverse micelle technique, to yield a water-soluble four-domain glycolipoprotein incorporating an average of 10 glycolipid chains per BSA molecule. The immunogenicity of this conjugate is under evaluation in mice.

Synthetic vaccine based on oligomers of ribitol-phosphate.

Polymers of ribitol phosphate have been found in several human pathogens as part of their capsular and cell wall structures. Vaccines based on the CPS, composed of ribose-ribitol-phosphate, of Haemophilus influenzae type b have been highly successful. We synthesized octa- and dodecamers of ribitol phosphate and covalently attached them to BSA at various saccharide-protein ratios. We are evaluating the conjugates' immunogenicity and cross-reactivity.

Definition of angular dependence of 1H-15N coupling constants in amino-sugar derivatives

Coxon

High-resolution nuclear magnetic resonance spectroscopy is a powerful method for analysis of the conformations, molecular structures, purity, and stereochemistry of carbohydrates, including monosaccharide, oligosaccharide, and polysaccharide types that are of prime interest in bacterial vaccine development. Part of our mission is to provide analytical NMR support as needed for current vaccine projects that involve the characterization of saccharide structure and purity; another is to conduct fundamental investigations of the application of measurements of NMR chemical shifts, nuclear spin-spin coupling constants, and nuclear Overhauser effects to the structural analysis of carbohydrates. We also characterize the molecular geometry of saccharides by molecular dynamics computations followed by energy minimization by molecular mechanics.

Amino sugars are common components of bacterial polysaccharides. As part of a program of vaccine development based on such polysaccharides, we are interested in extending the use of nitrogen NMR parameters for the structural, stereochemical, and conformational analysis of these materials. In recent studies of amino sugar derivatives, we characterized the NH- to ND-induced isotope effects on the chemical shifts of 13C nuclei surrounding the nitrogen atom. The high sensitivity of an NMR cryoprobe allowed the rapid acquisition of well-resolved one- and two-dimensional NMR spectra before significant anomerization of the N-acetyl-amino-deoxy sugars had occurred, thus facilitating their spectral assignment.

In our current work, we measured the 15N-1H coupling constants of various derivatives of amino sugars, using the inverse-detected, heteronuclear single quantum, multiple-bond correlation (HSQMBC) technique. We compared the obtained values with those measured by the more traditional procedure of synthesizing highly enriched 15N-labeled amino-sugar derivatives and analyzing their 1H NMR spectra. The HSQMBC method is highly selective, in this instance filtering out all non-15N NMR interactions, thereby yielding simplified NMR spectra. The enhanced sensitivity of a 1H/13C/15N inverse NMR cryoprobe facilitated acquisition of the HSQMBC spectra from reasonably small quantities of materials (for example, 30 mg/0.5 mL) with 15N at natural abundance.

We studied several bicyclic amino sugar models of fixed or known geometry, including a series of methyl 2- and 3-amino-4,6-O-benzylidene-deoxy-alpha-D-hexopyranosides in chair or skew boat conformations as well as methyl 2,6-anhydro-3-deoxy-3-phthalimido-alpha-D-mannopyranoside in a locked, almost classical boat conformation. We determined the major conformational features of these molecules from 1H-1H NMR coupling constants measured by complete assignment of the 1H NMR spectra of the amino derivatives. We correlated the magnitudes of the vicinal 1H-15N coupling constants with the geometry of the coupled nuclei in the conformations of the amino-sugar models, as determined by molecular dynamics/mechanics computations. We investigated various methods for analysis of the HSQMBC spectra, finding the most convenient to be "NMR multiplet total width subtraction" based on the fact that the total width of first-order NMR multiplets is equal to the sum of their coupling constants.

Non-linear regression of the vicinal 1H-15N coupling constants to the HCCN dihedral angles determined by molecular dynamics led to the definition of a new Karplus equation: 3JHCCN = 3.1 cos2 phi = 0.6 cos phi + 0.4, which describes the dependence of the vicinal, 3JHCCN coupling constant on the HCCN dihedral angle phi in amino-sugar derivatives. We expect the equation to be applicable in NMR analysis of the stereochemistry of amino-sugar components of nitrogen-containing saccharides and aminoglycoside antibiotics.

Verification of the structures of bacterial polysaccharides by NMR spectroscopy

Coxon, Jin, Robbins; in collaboration with Bohach, Deobald, Freedberg, Norris, Shiloach

We analyzed polysaccharides of interest by one or more of a variety of NMR methods, including one-dimensional 1H NMR, two-dimensional correlation spectroscopy (COSY), and three-dimensional total correlation spectroscopy (TOCSY) for assignment of 1H spectra and one-dimensional 13C NMR, 1H coupled and decoupled two-dimensional heteronuclear single quantum correlation (HSQC), and two-dimensional heteronuclear multiple bond correlation (HMBC) for the elucidation of 13C NMR spectra.

Neisseria meningitidis W135.

We used 1H NMR spectroscopy to analyze preparations of native and de-O-acetylated capsular polysaccharide from W135 CPS for N-acetyl and O-acetyl content. Quantitative integration of the N-acetyl and O-acetyl methyl signals of the 7-O-acetyl- and 9-O-acetyl-N-acetylneuraminic acid components indicated that the total O-acetyl content of the CPS was 1.1-1.2 mole percent, i.e., close to the detection limit of the spectrometer.

Haemophilus influenzae A.

We analyzed preparations of the native and purified CPS by one-dimensional 1H NMR, one-dimensional 13C NMR, two-dimensional COSY, two-dimensional TOCSY, 1H coupled and decoupled two-dimensional HSQC, and two-dimensional HMBC. The strongest evidence for the polyphosphate structure of the CPS was apparent in the 13C NMR spectrum, which displayed five 13C-31P coupling constants in the appropriate 13C resonances. NMR also indicated that the purity of the preparations was high.

¹ Oak Ridge Senior Fellow Program at NIH

COLLABORATORS

Amina Ahmed, MD, Carolina Medical Center, Charlotte, NC
Glen D. Armstrong, PhD, University of Calgary, Calgary, Canada
Shai Ashkenazi, MD, Schneider Children's Hospital, Petah Tikva, Israel
Joseph Bellanti, MD, Georgetown University Medical Center, Washington, DC
Gregory Bohach, PhD, University of Idaho, Moscow, ID
Dianjun Cao, PhD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Arturo Casadevall, MD, PhD, Albert Einstein College of Medicine, New York, NY
Bo Claesson, MD, Sahlgrenska University Hospital, Göteborg, Sweden
John Clements, PhD, Tulane University Health Sciences Center, New Orleans, LA
Daron I. Freedberg, PhD, Center for Biologies Evaluation and Research, FDA, Bethesda, MD
Aharona Glatman-Freedman, MD, Albert Einstein College of Medicine, New York, NY
Magnus Gottfredsson, MD, Lanspitali University Hospital, Reykjavik, Iceland
Tyra Grove Krause, PhD, Statens Serum Institut, Copenhagen, Denmark
Lars Hanson, MD, Göteborgs Universitet, Göteborg, Sweden
Peter Hoogerhout, PhD, Nederlands Vaccin Instituut, Bilthoven, Netherlands
Yasutaka Hoshino, DVM, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Albert Z. Kapikian, MD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Dennis J. Kopecko, PhD, Division of Bacterial Products, CBER, FDA, Bethesda, MD
Teresa Lagergard, PhD, Göteborgs Universitet, Göteborg, Sweden
Stephen H. Leppla, PhD, Laboratory of Bacterial Diseases, NIAID, Bethesda, MD
Louis Miller, MD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
Mark Miller, MD, Fogarty International Center, NIH, Bethesda, MD
Sophie E. Moore, PhD, London School of Hygiene and Tropical Medicine, London, UK
Nga Y. Nguyen, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Scott E. Norris, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Ruth Nussenzweig, MD, PhD, New York University School of Medicine, New York, NY
Justen H. Passwell, MD, Chaim Sheba Medical Center, Tel-Hashomer, Israel
Andrew M. Prentice, MD, London School of Hygiene and Tropical Medicine, London, UK
Donald C. Robertson, PhD, Kansas State University College of Veterinary Medicine, Manhattan, KS
Joseph Shiloach, PhD, Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD
Darrell Singer, MPH, Medical Corps, Walter Reed Army Institute of Research, Washington, DC
Evgeny Vinogradov, PhD, Institute for Biological Sciences, National Research Council, Ottawa, Canada

For further information, contact schneerr@exchange.nih.gov.