Development of Vaccines Against Bacterial Diseases Especially in Children

John B. Robbins, MD, Co-Chief, Program in Developmental and Molecular Immunity
Rachel Schneerson, MD, Co-Chief, Program in Developmental and Molecular Immunity; Head, Section on Bacterial Disease Pathogenesis and Immunity
Audrey L. Stone, PhD, Senior Investigator
Chiayung Chu, MD, Staff Scientist
Vince Pozsgay, PhD, Staff Scientist
Shousun C. Szu, PhD, Staff Scientist
Feng-Ying (Kimi) Lin, MD, MPH, Medical Officer
Bruce Coxon, PhD, DSc, Senior Research Fellow
Darrell Liu, PhD, Senior Research Fellow
Victor C. Nelson, PhD, Senior Research Fellow
Peter Ftacek, PhD, Research Fellow
Zhigang Jin, MD, Research Fellow
Joanna Kubler-Kielb, PhD, Research Fellow
Zuzanna Biesova, PhD, Postdoctoral Fellow
Patrick Claude, PhD, Postdoctoral Fellow
Andres Dulcey, PhD, Postdoctoral Fellow
Ariel Ginzberg, PhD, Postdoctoral Fellow
Paul Santacroce, PhD, Postdoctoral Fellow
C. Goran Ekborg, PhD, Oak Ridge Fellow
Jerry Keith, PhD, Oak Ridge Senior Fellow
Arthur B. Karpas, PhD, Charles River Adjunct Investigator
Zhongdong Dai, MD, Adjunct Investigator
Yanping Wu, MD, Adjunct Investigator
Jianping Li, MD, Guest Researcher
Genene Beyene, BScEng, Charles River Research Assistant
Chunyan Guo, BSc, Charles River Research Assistant
Elizabeth Ogbonna, BSc, Charles River Research Assistant
Galina Kazanina, PhD, Senior Research Assistant
Fathy D. Majadly, BSc, Senior Research Assistant
Steven W. Hunt, BSc, Research Assistant
Christopher P. Mocca, MS, Research Assistant
loc B. Trinh, BSc, Research Assistant
Faye C. Chen, MS, Technician
Erica N. Turner, BSc, Postbaccalaureate Fellow

To achieve our overall objective of developing vaccines for infectious diseases, especially those of children, we study, discover, and re-evaluate clinical, epidemiologic, and immunologic data. We evaluate investigational vaccines suitable for clinical investigation, first, in experimental animals and then for submission to the Institutional Review Board and the FDA for evaluation of safety and immunogenicity in adults, children, and infants, and, finally, of efficacy.

Cross-reacting polysaccharides (H. influenzae types a and b and B. pumilus)

Robbins, Kubler-Kielb, Schneerson, Pozsgay, Chu; in collaboration with Carlone, Miller M, Romero-Steiner, Shiloach

Herd immunity followed widespread use of the Hib conjugate vaccine, and the near-elimination of Hib led to speculation that other Haemophilus influenzae (Hi) types may emerge as causes of meningitis. For example, in Brazil, the incidence of Hib meningitis decreased by 69 percent during the first year after initiation of Hib conjugate immunization while the incidence of Hia meningitis increased 8-fold. The Netherlands Reference Laboratory reported that type a was observed only in children younger than 4 years old. Our goal is to provide a vaccine against Hi type a, which is the second most prevalent type of Hi causing invasive diseases; it is especially common among certain populations of children. A second goal is to investigate the feasibility of providing a single vaccine to several pathogens. We have elucidated the structures of the six capsular polysaccharide (CP) types, a, b, c, d, e, and f of Hi (systemic infections were attributable mostly to H. influenzae type b [Hib]). A critical level of serum IgG anti–Hib CP confers type-specific immunity by complement-mediated bacteriolysis.

Properties of Hia and Hib CP.

Hi may be classified into three virulence groups of two that are related to their CP structures. Types a and b, which are the most virulent, are composed of a neutral sugar, an alcohol (ribitol), and a phosphodiester; types c and f are composed of an N-acetylated aminosugar, a monosaccharide, and a phosphodiester; and (3) types d and e have a repeat unit of an N-acetylglucosamine and N-acetylmannosamineuronic acid. As compared with the other four types, Hib and Hia exhibit greater resistance to the bactericidal effects of complement alone. Challenge of infant rats showed that the 50 percent infective dose (ID50) for bacteremia of both Hib and Hia was several logs lower than that of the other types. Intranasal challenge of infant rats with type b or type a resulted in 55 to 90 percent bacteremia with type b and 35 percent with type a. The other types were not invasive.

Invasive diseases caused by Hia.

Rates of Hia disease have remained constant in the United States despite Hib vaccination. From 1998 to 2002, the Emerging Infections Program of the CDC conducted active laboratory- and population-based surveillance for Hi disease in data from nine sites with approximately 35 million people. Seventeen of 1,743 invasive isolates were Hia, which is an important cause of meningitis in certain populations such as White Mountain Apache Indian children—with an annual incidence of 254 cases/100,000 children of Hia meningitis. Hammitt et al. (Pediatr Infect Dis J 2005;24:453) reported an outbreak of invasive Hia disease among Native Alaska infants. During a 6-month period in two proximate villages, five Hia cases were documented. In Brazil, an incidence of Hia meningitis of 0.16 cases/100,000 person-years was reported. South Africa and native populations in Manitoba Canada also report Hia meningitis in children.

Hia vaccine.

The number of Hia cases is too low for a randomized, double-blinded, controlled trial. Yet, precedent exists for adding types within a species to a vaccine without evidence for efficacy. For example, the licensing of several pneumococcal types, including meningococcal groups Y and W135 and poliovirus type 2, was based on their safety and immunogenicity. The structural, experimental, and clinical properties of Hia CP closely resemble those of type b, and the increasing number of reports of Hia-invasive disease suggests that development of an Hia conjugate is warranted. Methods for conjugating type b CP to a protein are applicable to Hia. D-1,5-ribitolphosphate is a constituent of the CPs of Hia and Hib. We reported that the cell wall polysaccharide (PS) of B. pumilus Sh18 contains a poly-1,5-ribitolphosphate as a major component and that antibodies induced in mice by such conjugate cross-reacted with both Hia and Hib. We synthesized polyribitolphosphate chains containing either 8 or 12 repeat units, with the terminal keto groups used for conjugation to aminooxylated bovine serum albumin (BSA or tetanus toxoid. We injected the conjugates into mice, three times at 2-week intervals at 2.5 g/mouse and obtained sera a week later. ELISA demonstrated antibodies to both Hia and Hib, with the octamer conjugate a better immunogen than the dodecamer conjugate. Some of the tested sera showed bactericidal activity against both type a and type b as roughly correlated with their ELISA values.

Jin Z, Romero-Steiner S, Carlone GM, Robbins JB, Schneerson R. Haemophilus influenzae type a infection and its prevention. Infect Immun 2007;75:2650-2654.

Shigellae

Schneerson, Robbins, Chu, Majadly, Mocca, Kubler-Kielb, Guo, Trinh; in collaboration with Ashkenazi, Chowers, Shiloach

Shigellosis is endemic throughout the world and hyperendemic in developing countries; its estimated annual incidence is about 200 million cases, with a mortality of about 650,000. Shigellosis is common among military recruits in field conditions, refugees, and patients in institutions. Shigella spp. are the most common cause of dysentery in children, leading to stunted growth. Vaccine development has been hindered because Shigella spp. are pathogens for and inhabitants of humans only. More than 100 years since the discovery of the causative organism, there is still no licensed vaccine against shigellosis. The WHO has declared the prevention of shigellosis a top priority, and it is the goal of our project.

Surface polysaccharides of pathogenic bacteria, including CPs or the O-specific polysaccharide (OSP) of lipopolysaccharides (LPS), function as both essential virulence factors and protective antigens. Covalent binding of these saccharides to medically useful proteins to form conjugates increases their immunogenicity and confers on them T cell dependence, making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non-toxic P. aeruginosa exoprotein A (rEPA) exhibited an efficacy of over 70 percent in young adults exposed to 6 to 14 percent attack rates. This conjugate and that of S. flexneri 2a bound to the succinylated exoprotein A (rEPA-succ) were safe and induced IgG antibodies to the homologous LPS in 1- to 4-year-olds. A randomized, blinded, Phase 3 study of the conjugates in 1- to 4-year-olds, with each conjugate serving as a control for the other, showed the vaccines to be safe. Immunogenicity and efficacy were age-related with little efficacy in 1- to 2-year-olds but with about 70 percent in 3- to 4-year-olds. As before, percentage increases in antibody levels were similar to those for adults, but the actual achieved levels were lower. Using Caco2 and HeLa cells, we studied the effect of the children’s (ages 1-4) immune sera and the IgG isolated from them on shigella invasion into epithelial intestinal cells. The sera inhibited shigella invasion in a type-specific manner. Pretreatment of the sera or of Caco-2 cells with O-SP abrogated these effects in a type-specific and dose-dependent manner.

To enhance the immunogenicity of the conjugates, we bound S. sonnei and S. flexneri 2a O-SPs to additional carrier proteins—tetanus toxoid (TT) and recombinant protective antigen (rPA)—and injected mice twice, with the same or a different carrier the second time. For S. flexneri 2a, the twice-injected TT conjugate induced the highest (over 4-fold) antibody levels. For S. sonnei, the TT conjugate followed by the rPA conjugate induced significantly higher levels than the other combinations.

We have shown that synthetic oligosaccharides of S. dysenteriae type 1 O-SP, bound by their reducing end to a carrier protein (sun configuration), induced significantly higher antibody levels than conjugates of the native O-SP bound to protein by several point attachments (lattice configuration). Synthesis of S. sonnei oligosaccharides was not successful. Therefore, we isolated low–molecular mass O-SP-core (O-SPC) fragments of the native O-SP and used them to bind to proteins by oxime linkages between the terminal Kdo residues of the reducing end core and the aminoxy linkers bound to either BSA or a non-toxic diphtheria toxin mutant. The coupling reaction was performed at a neutral pH, at room temperature, and in a short time. Levels of anti–S. sonnei LPS IgG induced by these conjugates in young outbred mice were significantly higher than those induced by the full-length O-SP conjugates.

The O-SP of Plesiomonas shigelloides O17 is structurally identical to that of S. sonnei, but only the core structure of S. sonnei has been published. We investigated the core structure of P. shigelloides O17, including its linkage to the O-SP, by nuclear magnetic resonance (NMR) and mass spectroscopy. On the basis of our studies, we assigned the following structure of the repeat unit:

                                         a-GalN-4-a-GalA-7-a-Hep-1-7 b-Gal-4
                                                                   |       |
          a-AltNAcA-3-b-FucNAc4N-4-b-GlcNAc-6-a-GlcN-4-a-GalA-3-a-Hep-3-a-Hep-5-Kdo
                                                                   |
                                                             b-Glc-2

Chowers Y, Kirschner J, Keller N, Barshack I, Bar-Meir S, Ashkenazi S, Schneerson R, Robbins J, Passwell JH. O-specific corrected polysaccharide conjugate vaccine-induced corrected antibodies prevent invasion of Shigella into Caco-2 cells and may be curative. Proc Natl Acad Sci USA 2007;104:2396-2401.
Kubler-Kielb J, Vinogradov E, Chu C, Schneerson R. O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a. Carbohydr Res2007;342:643-647.

Bordetellae spp.

Kubler-Kielb, Ginzberg, Schneerson, Guo, Mocca; in collaboration with Lagergard, Vinogradov

The major goal of this project is to investigate the structural characteristics of LPS and lipooligosaccharides (LOS) of human pathogens and use the information gained to bind the detoxified O-SP to carrier proteins in order to develop experimental vaccines against such pathogens. To this end, we are developing conjugation methods of wide applicability; our secondary goal is to develop potential veterinary vaccines.

Bordetellae are Gram-negative bacilli that cause respiratory tract infections in mammals and birds. B. pertussis, B. parapertussis, and B. bronchiseptica are all clinically important. B. pertussis vaccines have succeeded in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action have not been established; there is no vaccine against B. parapertussis. Working from the concept that immunity to non-capsulated Gram-negative bacteria may be conferred by serum IgG anti–LPS, we studied chemical, serological, and immunological properties of the O-SP, obtained by different degradation procedures, from B. bronchiseptica and B. parapertussis obtained by different degradation procedures. Based on their non-reducing end saccharide structure, we identified one type of B. parapertussis O-SP and two types of B. bronchiseptica O-SP with no cross-reaction between the two B. bronchiseptica types. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SPs. For purposes of binding to an amino-oxylated protein, we prepared conjugates of B. bronchiseptica and B. parapertussis O-SP by using either the Kdo residue exposed by mild acid hydrolysis of the LPS or the core glucosamine residue exposed by deamination of the LPS. We carried out both coupling methods at a neutral pH, at room temperature, and in a short time. All conjugates injected into mice as saline solutions at a fraction of an estimated human dose induced antibodies to the homologous O-SP. These methods are applicable to the preparation of LPS-based vaccines against other Gram-negative bacteria.

Probably owing to the absence of the direct bactericidal effect of anti-toxin antibodies, the protection afforded by licensed subunit pertussis vaccines is incomplete on an individual basis. However, herd immunity, which occurs with wide vaccine usage, provides almost complete protection. Nonetheless, an additional vaccine component inducing bactericidal antibodies such as anti-LPS could increase vaccine efficacy on an individual basis. To that end, we isolated and analyzed B. pertussis and B. bronchiseptica LPS. B. pertussis expresses only the core region saccharide (OS), which is composed of 12 sugars. B. bronchiseptica, which is easier to culture and produces higher yields, expresses LPS with a core structure almost identical to that of B. pertussis, although we observed small variations; (1) the methylation of Fuc4Nme—100 percent in B. pertussis—is only 50 percent in B. bronchiseptica, and (2) Hep is phosphorylated at about 30 percent in B. bronchiseptica but not in B. pertussis. B. bronchiseptica LPS is mostly further substituted by O-specific chains. For this study, we used only a free core fraction with no O-SP. We prepared conjugates of both B. pertussis OS and B. bronchiseptica core by reacting their reducing-end Kdo moieties with an amino-oxy linker bound to BSA. Both conjugates incorporated an average of 10 saccharide chains per BSA molecule, both reacted with anti–B. pertussis and anti–BSA sera with a line of identity, and both were immunogenic in mice, inducing similar antibody levels as those measured by ELISA.

Kubler-Kielb J, Vinogradov E, Ben-Menachem G, Pozsgay V, Robbins JB, Schneerson R. Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates. Vaccine 2008;26:3587-3593.
Robbins JB, Schneerson R, Keith JM, Shiloach J, Miller M, Trollors B. The rise in pertussis cases urges replacement of chemically-inactivated with genetically-inactivated toxoid for DTP. Vaccine 2007;25:2811-2816.

Vaccine development for Group B Neisseria meningitidis and Escherichia coli K1

Robbins, Schneerson, Lin; in collaboration with Gottfredsson, Grove Krause, Howitz, Miller M, Mølbak

Vaccine development against Group B Neisseria meningitidis (GBM) meningitis is complicated by the pathogen’s capsular polysaccharide (PSA), an alpha 2-8-linked polysialic acid that is identical to the surface saccharide of the K1 capsular polysaccharide of E. coli as well as to host structures, especially during the host’s development. Despite effective antibiotic and supportive therapy, the mortality and morbidity of systemic infections, especially as a result of meningitis caused by GBM and Escherichia coli K1, remain unacceptably high. GBM continues to cause epidemics and outbreaks throughout the world, and E. coli K1 is a major cause of neonatal meningitis and kidney infections. With the goal of eventually providing a vaccine against these organisms, we examined evidence or the lack thereof for an association between PSA antibodies and autoimmune disease or effects on fetuses.

Meningococci are classified into serogroups according to their CPs. Of the 13 reported CP groups, 5 (A, B, C, W135, and Y) cause almost all meningococcal disease. As essential virulence factors, the five groups inhibit the protective actions of complement; in addition, they function as protective antigens as evidenced by the fact that a critical level of serum IgG CP antibodies specifically induces both complement-mediated lysis of groups A, C, W135, and Y and opsonophagocytic killing of group GBM. Effective CP-based vaccines exist for groups A, C, W135, and Y, but none exists for GBM or E. coli K1. GBM causes more infections in infants and young children than do menigococci of groups A, C, W135, or Y.

Although PSA antibodies bind to many fetal and adult tissues in vitro, there is no evidence for in vivo binding or associated pathology. We have therefore focused on developing vaccines that use non-capsular antigens, including outer membrane proteins, lipopolysaccharides, iron-binding proteins, and other antigens identified by examining the organism’s DNA. Many of these antigens are polymorphic, heterogeneous, and subject to antigenic variation and may not be representative of all GBM. Furthermore, none will be useful against E. coli K1. Based on the performance of the Haemophilus influenzae type b, Salmonella typhi (Vi), pneumococcal, and group C meningococcal (GCM) vaccines, we developed a PSA conjugate that induced protective levels of serum IgG anti–PSA. It is simple to produce and easy to standardize and should be close to 100 percent effective at all ages. We have confirmed its performance in laboratory mice.

Few studies have compared severity of infection and outcome among meningococcal serogroups, and published studies of the sequelae of meningococcal meningitis patients make no mention of autoimmune diseases, such as Guillain-Barré syndrome, multiple sclerosis, and so forth. For mortality or nearly every category of sequela associated with GBM meningitis, our review shows rates that are equal to or lower than those associated with other meningococcal serogroups. In the absence of epidemiological or clinical evidence to associate pathology with PSA antibodies, we conducted a retrospective cohort study of meningococcal patients to examine evidence for autoimmunity. The entire Danish population constituted our study cohort of 7,467,001 individuals followed for autoimmune disease between 1977 and 2004. GBM meningitis was diagnosed in 2,984 individuals, and the control population was 914 patients with GCM meningitis. Ratios of incidence rates of autoimmune diseases provided a measure of relative risk. Patients with GBM meningitis, either in comparison with people with group C meningococcal meningitis or those with no history of meningococcal meningitis, exhibited no increased risk of autoimmune diseases. We also studied this cohort for possible increased risk of preterm or stillbirth to women with previous GBM disease and whether their first-born children were at increased risk for birth defects. We found no such associations. Our findings suggest that systemic infection with GBM is not associated with autoimmune diseases or with immunoreactive diseases that may affect the health of offspring for up to 31 years after meningococcal disease.

In a Phase 1 study, 100 healthy adults will be injected with our PSA conjugate starting in early 2009. We expect to complete the study by the end of 2009.

Howitz M, Grove Krause T, Brunbjerg Simonsen J, Hoffmann S, Frisch M, Munk Nielsen N, Robbins JB, Schneerson RS, Miller MA, Mølbak K. Lack of association between group B meningococcal disease and autoimmune disease. Clin Infect Dis 2007;45:1327-34.
Howitz MF, Simonsen J, Krause TG, Robbins JB, Schneerson R, Mølbak K, Miller MM. Risk of adverse birth outcome after group B meningococcal disease: results from Danish National Cohort. Pediatr Infect Dis J, in press.

Peptide-protein conjugate vaccines

Schneerson, Robbins, Kubler-Kielb, Keith, Biesova, Lin, Liu, Wu, Majadly, Mocca, Guo; in collaboration with Bellanti, Leppla, Miller L, Miller M, Shiloach, Singer

Bacillus anthracis.

B. anthracis, a cause of lethal human infection with potential for bioterrorism, 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, Edema Factor, and Protective Antigen (PA). The capsule is composed of poly-d-gamma-glutamic acid (PGA). By itself, the capsule is non-immunogenic and its protective effect is not clear. Anthrax is transmitted indirectly, via spores, of different structure and composition than the vegetative organism. The PA-based licensed vaccine is safe and protective under normal conditions, but enhancement of its immunogenicity may be needed in the event of bioterrorism. We sought to induce capsular and anti-spore antibodies as a potential means to expand the immunity conferred by the available anthrax vaccines. We isolated a recombinant PA from an uncapsulated strain, several formaldehydetreated and/or alum-adsorbed formulations of which were immunogenic in mice. The formulations were safe in adult volunteers, and local and systemic reactions were rare and minor. We are now analyzing serum IgG anti–PA levels.

For comparison, we measured serum IgG anti–PA in 246 sera of recruits injected with the Anthrax Vaccine Adsorbed (AVA) and then analyzed paired sera by ELISA; the sera are stored in the U.S. Department of Defense Serum Repository. Serum conversion rates of greater than or equal to a 4-fold increase in antibody levels were pre-3rd–post-3rd 85.3 percent, pre-4th–post-4th 67.9 percent, and pre-6th–post-6th 45 percent. Geometric mean (GM) levels of all individuals were 59.9 µg per mL following the third injection, 157.4 µg per mL following the fourth injection, and 277 µg per mL following the sixth injection.

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 PGA and the proteins with active groups at the C- or N-termini yielded conjugates immunogenic in mice, with no statistically different responses to the conjugates. The induced antibodies were opsonophagocytic. Peptides 10- to 20-mer long and 10 to 15 mole PGA per mole protein were the most immunogenic. Dose-response experiments of an rPA -PGA conjugate, using between 0.31 and 20 µg PGA per mouse, showed 1.25 µg to be optimal for a PGA response while PA antibody levels increased with higher immunizing dosages. The use of alum increased PA antibody levels with little effect on anti–PGA levels. We also immunized two chimpanzees, one with PA-PGA and the other with TT-PGA, with the goal of preparing humanized monoclonal antibodies to PGA. Both responded with antibodies to both vaccine components. The chimpanzee injected with TT-PGA had higher anti–PGA levels. We prepared PGA-specific IgG1 and IgG3.

The glycosyl part of the glycoprotein BclA (collagen-like protein of B. anthracis) is an oligosaccharide composed of 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucose (referred to as anthrose) and three rhamnose residues. We found structures similar to anthrose in the sidechain of the CP of Shewanella spp. MR-4: 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-dglucose. Under certain growth conditions, the bacteria produce a variant CP lacking one methyl group on the hydroxybutyrate: 4-(3-hydroxybutanamido)-4,6-dideoxy-d-glucose. In contrast to anthrose, neither of the Shewanella CPs is 2-O-methylated. Both Shewanella CP variants reacted with anti–B. anthracis spore sera. We also found structures similar to anthrose in the flagellae of Pseudomonas syringae, which are reportedly glycosylated with a similar terminal saccharide: 4-(3-hydroxybutanamido)-4,6-dideoxy-2-O-methyl-d-glucose. In a fluorescence microscopy assay, sera produced by immunization with Shewanella or P. syringae cells bound to B. anthracis spores but not to B. cereus spores. Protein conjugates of the two variants of Shewanella CP induced antibodies that bound to both Shewanella CP variants, as judged by ELISA, and to B. anthracis spores, as detected by fluorescence microscopy. We propose the use of Shewanella CP conjugates as a component of an anthrax vaccine. We also elucidated the structure of the Shewanella MR-4 LPS carbohydrate backbone but found no anthrose-like sugar, confirming the presence of such a sugar in the CP only.

Plasmodium falciparum.

Malaria, a leading cause of morbidity and mortality globally, especially in children, is estimated to cause over a 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. We therefore set out to develop vaccine candidates. The most studied experimental malaria vaccines are the circumsporozoite protein (CSP), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. The vaccines have been shown to be safe and immunogenic, but their protection is poor and of limited duration even when administered with adjuvants. We used two approaches to provide experimental malaria vaccines. The first was directed to the asexual, human-host stage of the parasite; we cloned the CSP into E. coli and, after isolation, evaluated several formulations, including adsorption onto alum, in young outbred mice. Based on findings from our studies with peptides of the B. anthracis capsule, we synthesized peptides of four to five repeat units of the four amino acids NANP (i.e., the four amino acids of the repeat unit frame shifted by one amino acid, yielding four configurations) and bound them to carrier proteins at different molar ratios and end groups. Injected into general purpose mice, all tested preparations induced high levels of antibodies that bound to circumsporozoites as demonstrated by immunofluorescence assay. Conjugates of NANP with the ookinete surface protein Pfs25 induced long-lasting antibodies to both vaccine components; 3-month serum levels were higher than at 1 week after the last injection. Alum-adsorbed CSP induced higher antibody levels than the non-adsorbed protein. We found that the terminal amino acid of the CSP-derived peptides was an important determinant and that NPNA-protein was the best immunogen.

The second approach was directed to the sexual, mosquito parasite stage in order to provide a transmission-blocking vaccine. We bound Pfs25—a low–molecular-mass protein that is non76 immunogenic by itself—to itself, or to carrier proteins by amide, hydrazone, or thioether linkages. Injected into mice, all conjugates were immunogenic with booster responses upon reinjection. Remarkably, the serum antibody levels at 3 and 7 months after immunization were higher than at 1 week after the last injection. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum further increased antibody levels. As measured by ELISA, transmission-blocking activity of immune sera correlated with antibody levels.

Kubler-Kielb J, Majadly F, Wu Y, Narum DL, Guo C, Miller LH, Shiloach J, Robbins JB, Schneerson R. Long-lasting and transmission-blocking activity of antibodies to Plasmodium falciparum elicited in mice by protein conjugates of Pfs25. Proc Natl Acad Sci USA 2007;104:293-298.
Kubler-Kielb J, Vinogradov E, Hu H, Leppla SH, Robbins JB, Schneerson R. Saccharides cross-reactive with Bacillus anthracis spore glycoprotein as an anthrax vaccine component. Proc Natl Acad Sci USA 2008;105:8709-8712.
Singer DE, Schneerson R, Bautista CT, Rubertone MV, Robbins JB, Taylor DN. Serum IgG antibody response to the protective antigen (PA) of Bacillus anthracis induced by anthrax vaccine adsorbed (AVA) among U.S. military personnel. Vaccine 2008;26:869-873.
Vinogradov E, Kubler-Kielb J, Korenevsky A. The structure of the carbohydrate backbone of the LPS from Shewanella spp. MR-4. Carbohydr Res 2008;343:2701-2705.

Protein and polysaccharide conjugate vaccines to enteric diseases

Szu, Schneerson, Lin, Ftacek, Hunt, Li, Nelson, Wu; in collaboration with Ahmed, Clements, Griffin, Kopecko, Moore, Robertson, Shiloach, Cao, Hoshino, Kapikian, Osterholm

Diarrheal diseases account for more than 3 million deaths annually. Diseases such as cholera are common in developing countries and are underreported. Few vaccines for enteric infections are licensed, and all have limitations. For example, the newly licensed rotavirus vaccines elicit only low levels of antibody in infants and may cause adverse reactions. Whole-cell vaccines for enteric fevers and for cholera were withdrawn from the market because of adverse reactions. Our goal is to prepare vaccines against enteric infections suitable for routine administration to infants and young children. We study investigational vaccines against bacteria such as Salmonella typhi, non-typhoidal Salmonellae, Escherichia coli (in particular E. coli O157), Vibrio cholerae, and Campylobacter jejuni and viruses such as rotavirus.

Our vaccines are designed to induce serum IgG that inactivates the inoculum. The protective antigens are proteins, such as toxins or capsid proteins, and polysaccharides, such as capsules or lipopolysaccharides. To enhance antigen immunogenicity, we conjugated polysaccharides and polypeptides to proteins. Surface polysaccharides of Gram-negative pathogens—capsules or LPSs—are essential virulence factors and protective antigens. Covalent binding to carrier proteins enhances the immunogenicity of polysaccharides. To serve as vaccines, LPSs must be detoxified and their O-SPs isolated and conjugated to proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and serve as carrier proteins.

The Vi capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine with limited efficacy in children under 5 years old. To provide a vaccine for younger children, we conjugated Vi to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A Phase 3 trial of Vi-rEPA in 11,600 Vietnamese 2- to 5-year-olds showed an efficacy of 89 percent at 47 months. In a Phase 2 trial, 301 infants were injected with Vi-rEPA concurrently with DPT at 2, 4, 6, and 12 months. Controls received Hib-TT plus DTP or DTP. No serious adverse reactions occurred. We will assay IgG anti–Vi levels in the infants and compare them with those elicited in 2- to 5-year-olds. We evaluated antibody persistence after vaccination with Vi-rEPA in adults and children and found that the GM IgG anti–Vi level of 18 adults 10 years after a single injection was 20-fold higher than the proposed protective level (3.52 EU), whereas the GM antibody level of 97 children injected twice at ages 2 to 5 years, 9 years after immunization, was only 2.2-fold higher than the proposed protective level. We observed no significant difference between Vi antibody levels in the latter group and in those that had only one injection (7.04 EU) or no injection (8.14 EU), reflecting a waning of anti–Vi in young children on one hand and the development of natural antibodies on the other. Four years after a single injection into 5- to 8-year-olds, the GM antibody level was 15.9 EU, significantly higher than in age-matched controls.

Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries. Our Phase 1 and 2 studies showed that SPA O-SP conjugated to TT was safe and immunogenic in adults, teenagers, and toddlers. To improve conjugate immunogenicity, our collaborator Dennis Kopecko replaced the SPA LPS chain-length elongation regulator gene wzz with that of E. coli K12. Several TT conjugates of the elongated O-SP elicited significantly higher levels of anti–LPS than those prepared with O-SP from the wild type.

Enterotoxigenic E. coli (ETEC) is the most common cause of diarrhea in developing countries and in travelers to such countries. ETEC disease is mediated by two exotoxins: heat-labile toxin (LT) and heat stable toxin (ST). LT is immunogenic in humans. ST is a polypeptide of 19 amino acids and is not immunogenic because of its small size. The exotoxins’ efficacy against infection has not been demonstrated in humans. LT 192, a mutant of reduced toxicity (provided by John Clements), elicited high levels of IgG anti–LT in mice. However, dosage-dependent swelling occurred at the injection site even after formalin detoxification, an adverse reaction that could be reduced by using a double mutant LT (mutation at amino acids 192 and 211) as an immunogen. The wild-type LT and the double mutant elicited similar IgG anti–LT levels in mice. We plan to undertake clinical studies with the double mutant. Several attempts at chemical synthesis of ST have failed owing to ST’s high number of disulfide bonds (three pairs). In collaboration with Donald Robertson, we purified ST from E. coli. We have prepared a conjugate of ST linked to bovine serum albumin and will evaluate its immunogenicity in mice.

Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that serum vibriocidal activity is directed toward the bacterium’s LPS. In a Phase 1 trial, O-SP conjugates elicited IgG anti–LPS with vibriocidal activity. To enhance immunogenicity, we devised a new scheme of conjugation by thiolating both the carrier protein and the O-SP with heterobifunctional reagents. Antibody levels induced in mice by these conjugates were significantly higher than those induced by the conjugate used in our Phase 1 study. We also synthesized a hexamer of V. cholerae O-SP. Conjugated to TT, the hexamer was antigenic. We plan to evaluate the immunogenicity of both the new bacterial O-SP conjugates and the synthetic saccharide conjugates.

E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. An O157 O-SP-rEPA conjugate, injected into 55 2- to 5-year-old children once or twice, was safe and immunogenic; 6 months after injection, 98 percent of the children had a greater than 4-fold increase in IgG anti–LPS over levels before immunization. The second injection did not induce a booster response. In collaboration with Patricia Griffin and Michael Osterholm, we developed a proposal to immunize children between 2 and 10 years of age in States with the highest incidence of E. coli O157 infection.

Rotavirus is the most common cause of infantile diarrhea worldwide. Two rotavirus vaccines are licensed—a live attenuated vaccine and a reassortant vaccine, both orally administered. We are designing a parenteral vaccine based on capsid proteins VP8 and VP7. We purified VP8 from baculovirus-infected insect cells (from Yasutaka Hoshino) and observed that mice injected with VP8 produced neutralizing antibodies against rotavirus of both homologous (P4) and heterologous (P8) serotypes. To increase the yield of VP8, we expressed its cDNA in E. coli. The recombinant VP8 elicited neutralizing antibodies to both P4 and P8 rotavirus serotypes.

Moore SE, Jalil F, Szu SC, Hahn-Zoric M, Prentice AM, Hanson LA. Revaccination does not improve an observed deficit in antibody responses in Pakistani adults born of a lower birth weight. Vaccine 2008;26:158-165.

Synthetic vaccines against human pathogenic bacteria

Pozsgay, Schneerson, Beyene, Claude, Dulcey, Ekborg, Santacroce

Using innovative glycochemical technologies, we design and synthesize oligosaccharide- and glycolipid-based conjugate vaccines for the prevention of human diseases. We develop high-yielding, mild, and experimentally simple methods for covalent conjugation of glycans to proteins. Human bacterial pathogens may have surface-exposed saccharides that serve as both virulence factors and protective antigens. Such saccharides include CPs, LPSs, and cell-wall polysaccharides varying in size and complexity. We are studying synthetic chemical approaches to bacterial surface–exposed saccharides that can be used to elicit serum IgG antibodies against such pathogens.

Synthetic glycoconjugates as experimental vaccines.

Shigella dysenteriae as acquired resistance to most available antibiotics. Evidence suggests that antibodies to the O-SP of the LPS of this bacterium may be protective. The O-SP consists of a tetrasaccharide repeating unit containing l-rhamnose, d-galactose, and N-acetyl-d-glucosamine units with the →3)-alpha-l-Rhap-(1→2)-alpha-d-Galp-(1→3)-alpha-d-GlNAcp-(1→3)-alpha-l-Rhap-(1→ linear tetrasaccharide structure. We have shown that covalent conjugates of bovine serum albumin to oligosaccharides corresponding to the O-SP in the 8- to 16-mer range elicit O-SP–specific antibodies in mice. We achieved the highest antibody levels when the non-reducing terminus was occupied by either a d-galactose or an N-acetyl-d-glucosamine residue, whereas oligosaccharides that contained an l-rhamnose residue at that terminus produced low levels of anti-saccharide antibodies. With this finding, we have redesigned our previous synthetic sequence and prepared a decamer and an undecamer containing the GlcNAc-Rha-Rha-Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha and the Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha structures, in amounts exceeding 300 mg. When using our oxime-based conjugation method, these oligosaccharides contain a linker at their reducing end for covalent attachment to a medically acceptable protein. We plan to evaluate the conjugate-induced anti–O-SP antibody levels in volunteers.

It has been proposed that Shigellae are descendants of E. coli and that E. coli O-148 is the precursor of S. dysenteriae type 1. Similar to S. dysenteriae type 1, E. coli O-148 causes diarrhea in children, soldiers, and travelers. The repeating unit of the O-SP in E. coli O-148 is almost identical to that of S. dysenteriae type 1; the only difference is that d-glucose replaces the galactose residue in the latter. To establish cross-reactivity between the two bacteria, we chemically synthesized oligosaccharide fragments of the O-SP of E. coli 148. These probes are composed of 1, 2, and 3 consecutive repeating units containing 4, 8, and 12 monosaccharide residues. The saccharide constructs also contain a spacer equipped with a keto moiety. Using our oxime-based conjugation technique, we have prepared human serum albumin conjugates containing up to an average of 28 oligosaccharide chains per protein molecule. We will investigate the immunogenicity of these conjugates to study cross-reactivity with the S. dysenteriae type 1 O-SP.

Synthetic vaccine against Borrelia burgdorferi.

Borrelia burgdorferi, the etiological agent of Lyme disease, expresses two major glycolipids on its surface. Termed BBGL-1 and BBGL-2, both constructs contain a galactose residue in addition to palmitoyl and oleoyl moieties. BBGL-1 also has a cholesterol unit, whereas BBGL-2 has a glycerol residue acylated by the palmitic and oleoic acids. The location of the two glycolipids on the bacterial surface makes them a suitable target for vaccine development. We have chemically synthesized BBGL-1 in both its native and bioconjugatable forms. Native BBGL-1 or its protein conjugate elicited low levels of anti–BBGL-1 antibodies in mice. We are now examining the immunogenicity of BBGL-2. First, we undertook its chemical synthesis. Starting from a commercially available, optically active glycerol derivative, we attached a fully protected galactose residue stereospecifically to the free hydroxyl of the glycerol moiety. Next, we removed the original protecting groups on the glycerol. We acylated the resulting diol regioselectively with palmitic acid followed by placement of the oleoyl moiety at the secondary hydroxyl group of the glycerol residue. Chemoselective removal of the protecting groups on the galactose moiety yielded BBGL-2. We will adapt this methodology for the synthesis of a conjugatable derivative of BBGL-2, which we will link to a protein in order to evaluate the feasibility of enhancing immunogenicity by conjugation to an immunogenic protein.

Kubler-Kielb J, Vinogradov E, Ben-Menachem G, Pozsgay V, Robbins JB, Schneerson R. Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates. Vaccine 2008;26:3587-3593.
Pozsgay V. Recent developments in synthetic oligosaccharide-based bacterial vaccines. Curr Top Med Chem 2008;8:126-140.
Pozsgay V, Kubler-Kielb J. Conjugation methods toward synthetic vaccines. In: Roy R, ed. Carbohydrate-based Vaccines. ACS Symposium Series. Oxford University Press, 2008;989:36-70.
Pozsgay V, Kubler-Kielb J. Synthesis of an experimental glycolipoprotein vaccine against Lyme disease. Carbohydr Res 2007;342:621-626.

Neonatal respiratory distress related to colonization with group B streptococci

Lin, Ogbonna, Turner; in collaboration with Curtis, Levine, Troendle, Yergey

Despite the decrease in the incidence of early-onset group B streptococcal (GBS) disease as a consequence of the widespread use of antibiotic prophylaxis during labor, GBS remains a leading cause of neonatal sepsis. We hypothesized that newborns colonized with GBS, especially newborns of penicillin-treated mothers, acquire bacterial phospholipids as a result of prophylaxis during labor, leading to pulmonary hypertension and respiratory distress. It has been shown that infusion of live or heat-killed GBS into sheep promptly induced pulmonary hypertension. The goal of our study was to examine clinical and epidemiologic data related to this potential association. Clinical features of early-onset GBS disease are similar to those of endotoxic shock, and a prominent symptom is respiratory distress, which may occur in newborns without a known GBS infection. Jerri Curtis and colleagues purified and identified phospholipids (cardiolipin and phosphatidylglycerol) from the GBS cell wall as pulmonary hypertensive compounds and observed that infusion of either cardiolipin or phosphatidylglycerol into neonatal lambs caused pulmonary hypertension. Others have demonstrated that exposure of Streptococcus mutans to penicillin induces a 15-fold increase in phospholipid release from the organism. Penicillin is one of the beta-lactam antibiotics, which inhibit the normal assembly of peptidoglycan of bacterial cell wall. Given the recommendation to treat newborns infected with GBS or their GBS-carrying mothers with penicillin or its derivatives, our findings raised the question of whether exposure of GBS to penicillin or to other lactam antibiotics in newborns and mothers induces a release of GBS phospholipids causing pulmonary hypertension respiratory distress in newborns.

Analysis of a data file from the NICHD multicenter GBS study found a possible association among GBS colonization, penicillin treatment, and respiratory distress in neonates, namely that nearly 9 percent of GBS-colonized newborns who did not develop early-onset GBS disease experienced respiratory distress within 48 hours of birth compared with 1 to 3 percent of non-colonized newborns. Furthermore, colonized newborns of penicillin-treated mothers were more than twice as likely to develop respiratory distress as GBS-carrying but untreated mothers.

Although the effect of cardiolipin on pulmonary hypertension and of penicillin on phospholipid release from S. mutans supports the role of GBS colonization and penicillin in respiratory distress in newborns, proof of these associations requires assay of GBS phospholipids in serum samples of newborns—with and without respiratory distress—born to GBS-colonized mothers treated or untreated with penicillin or other beta-lactam antibiotics during labor. We are developing such an assay in collaboration with Alfred Yergey. A prospective study aimed at relating serum GBS phospholipid levels to the occurrence of respiratory distress in newborns of GBS-colonized mothers is under way at two U.S. academic centers: Baylor College of Medicine, Houston, Texas; and Children’s Hospital and Research Center, Oakland, California. Our sample size estimate suggests a need for 1,600 colonized mothers who are at least 32 weeks’ gestation at delivery. We use a semiquantitative culture method to determine the degree of colonization in the mothers. We are assembling three groups of GBS-colonized mothers: untreated, beta-lactam antibiotic–treated, and treated with non–beta-lactam antibiotics. Maternal and cord blood samples are being collected for the cardiolipin assay, and all newborns are monitored for signs of respiratory distress. We are assembling two groups of newborns—with and without respiratory distress.

Design, synthesis, and testing of recombinant proteins for influenza vaccines

Keith, Robbins, Biesova, Schneerson, Trinh; in collaboration with Miller M, Shiloach

Highly pathogenic H5N1 avian influenza A virus has the potential to cause the next human influenza pandemic. To avert a world health crisis, innovative methods of rapidly producing sufficient quantities of human vaccine must be explored. Early each year, circulating human influenza virus strains are selected to be included in the vaccine formulation for the upcoming season. During the next 9 months, vaccine manufacturers produce the vaccine by growing the selected virus strains in embryonated chicken eggs. This vaccine production platform, with a maximum capacity of 300 million doses, lacks the capacity for rapid production of the 2 to 3 billion vaccine doses that would be required to prevent a worldwide avian flu pandemic. The essential immunogen in all influenza vaccines is the immunodominant hemagglutinin protein (HA). FDA mandates a minimum of 15 µg HA per dose. Based on the immunological diversity of HA, the WHO classified avian influenza viruses into three clades and several subclades and has encouraged vaccine developers to produce human vaccines effective against these virus clades. Our approach to this challenge is to use recombinant DNA technology, cellular protein expression systems, and chemical conjugation methods to produce unique protein antigens as influenza vaccine candidates.

We have developed a protocol for rapid production of the HA of H5N1 avian pandemic influenza viruses. The procedure does not require access to the pandemic influenza virus strains—select agent regulations, USDA control measures, and biosafety level constraints restrict our ability to work with the pathogens. Using only DNA sequences from the CDC/WHO pandemic influenza virus database, we chemically synthesized genes de novo in a procedure optimized for protein expression in E. coli. The procedure can reduce the response time required to produce a vaccine to less than 1 month compared with the 6 to 8 months needed for the traditional egg-based preparation of influenza vaccines. In addition to this new de novo gene protocol, we have developed cloning protocols that use influenza viruses as templates for reverse transcription-polymerase chain reaction gene amplification. With these procedures and protocols, we have constructed bacterial seed clones/cultures, used them to produce pilot plant quantities of recombinant HA proteins, and have now developed standardized downstream processing protocols for protein extraction, purification, refolding, and vaccine formulation. We have produced recombinant HA immunogens from four representative influenza A viruses: H3N2 A/California/7/2004 (standard control from human 2006 influenza vaccine); H5N1 A/Vietnam/1203/2004 Clade 1; H5N1 A/Indonesia/5/2005 Clade 2, subclade 1; and H5N1 A/Bar-headed Goose/Qinghai/1A/2005 Clade 2, subclade 2.

We designed the recombinant protein constructs to represent the mature configuration of HA, with the amino acid domain spanning the viral membrane deleted from the carboxyl terminal. We then replaced the domain with a Gly3X-His6X tag to facilitate purification of the expressed protein by Ni-ion chelating chromatography. We produced recombinant HA (rHA) from four representative virus strains in either BL21(DE3) or Rosetta 2(DE3) strains of E. coli and purified it from isolated inclusion bodies by urea solubilization of the inclusion body protein and by Ni-ion column chromatography. Using rapid dilution into refolding buffer, extensive dialysis, and spin-filter concentration, we further processed the purified rHA protein derived from the A/Vietnam/1203/2004 and A/Indonesia/5/2005 H5N1 influenza viruses for use in vaccine formulation and immunization studies. We formulated various vaccine candidates by adsorbing the rHA onto alum, treating the protein with formalin, or both. Injected into young mice three times (2.5 to 5µg per mouse), rHA induced antibodies with hemagglutination inhibition titers of 40 and 80 or higher, suggesting that rHA could induce protective immune responses against influenza virus infection (FDA guidelines require a minimum titer of 40). Our preliminary data suggest that the alum-absorbed rHA vaccine, produced in just 4 weeks, can fulfill FDA requirements. We plan to conduct antibody neutralization experiments based on plaque reduction analysis and inhibition of viral replication within a unique tissue culture cell line.

Recent reports in the literature suggest that antibodies to the exposed N-terminal of 23 amino acids (M2e) of the mature matrix 2 protein (M2) may ameliorate disease symptoms. The M2 protein provides an ion-channel through the viral membrane and is recognized as a target for prophylaxis and treatment with the antiviral drug amantadine. Unlike the virion’s surface proteins HA and neuraminidase, which are subject to constant genetic drift and shift, the M2 protein is highly conserved probably because its protected location within the viral membrane prevents a strong host immune response. However, recent studies showed that when the exposed 23–amino acid M2e peptide was genetically fused to the N-terminus of hepatitis B virus core particles as a carrier, the chimeric protein conferred complete protection against a lethal heterologous influenza virus challenge in a mouse model. We bound a synthetic M2e peptide to a genetically detoxified diphtheria toxin (DT-H21G) via thioether linkages. MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) analyses showed an average of seven chains of M2e per DT molecule. We injected the conjugate, in aqueous form or alum-adsorbed, into mice at 2.5 µg peptide, two or three times, 2 weeks apart and collected sera 1 week after the last injection. ELISA showed high anti–M2e levels after the third injection, with or without alum. We also observed an antibody response against the DT(H21G) carrier. Our preliminary results suggest that this candidate vaccine may induce immunity against heterologous strains of influenza A virus.

Modulation of protein and cell functions by heparin/heparan sulfate and mimetics

Stone, Chen, Kazanina; in collaboration with McMahon, Horne, Lewis, Longas

The rate of HIV infection is rising, now reaching over 1 million cases in the United States and 50 times that globally, with long-term control threatened by drug toxicity, the emergence of multidrug resistant strains, and treatment options limited mostly to protease and reverse transcriptase inhibitors. Thus, inhibitors against viral components that play other roles in HIV-1 attack are critically needed.

Found in cell membranes, heparin and heparan sulfates (H/HS) constitute a class of highly sulfated glycocaminoglycan biopolymers based on the specificity of their optically active structures (Stone, Biopolymers 1963;2 and 1964;3). These polysaccharide polymers occur as modulatory receptor systems throughout the body. Their diverse sulfation provides H/HS with unique sugar sequences (S-oligoS), accounting for their ability to modulate diverse proteins and biological systems in cell growth, inflammation, development, and microbial infections. Current structural considerations indicate that the S-OligoS structure contains a tetrasaccharide motif of three xyloses and a glucuronic acid as a branch on the xylan chain, i.e., -d-glucuronyl-alpha 1,2 beta 1,4 d-(xylyl)3 with up to one-third of the GlcA O-methylated. The sugars are about 90 percent sulfated, yielding a high negative charge density. Such a motif could accommodate the subtle variations in sulfate geometries, potentially providing for multifunctional mimicry of the heparins.

Owing to the similarity of the physicochemical properties of H/HS chains and absent cognate ligands, libraries of unique S-oligoS are not obtainable for research. We obtained a heparin-mimetic library that is based on the structure-function model for the antithrombin-dependent anticoagulant heparin (Stone et al., Proc Natl Acad Sci USA 1982;79:7190) and that comprises S-oligoS that mimic unfractionated heparin in its biological actions. We demonstrated that in vitro inhibition of both HIV-1 cytotpathology and syncytium formation are governed by structural specificity and separable from the anticoagulant S-oligoS—essential indicators for useful drug development (Stone et al., Glycoconjugate J 1998;15:697). We devised a procedure to obtain a clinical preparation of a highly active inhibitor of HIV-1 virus fusion free from antithrombin toxicity (SOLIS). Our clinical preparation of SOLIS for a Phase I safety trial is ongoing. Heparin has long been known to affect conformation of proteins, thus modulating their biological function. Given that conformational changes in bound gp41 are required for fusion progression, inhibition of this process is a possible mechanism of action of heparin and SOLIS.

Abu-Raddad et al. (Science 2006;314:1603) “estimated that, since 1980, the disease interaction may have been responsible for 8,500 excess HIV infections and 980,000 excess malaria episodes” in a selected Kenyan population. Co-treatment of malaria and HIV/AIDS is feasible because of the similarity of the respective S-oligoS inhibitors. We identified another component (Cp11) as a potential antimalaria drug (see below). We posited that SOLIS and Cp11 would be physicochemically compatible for combined treatment formulation and thus studied the effect of co-treatment in vitro. We investigated whether the addition of increasing amounts of Cp11 (relatively inactive against HIV-1) to the standard SOLIS doses would affect the capacity of SOLIS to protect cells in a standard formazan cytotoxicity protection assay. Results indicated that the presence of Cp11, the antimalarial S-oligoS, diminished SOLIS’s inhibitory effect in vitro, which would cause a significant mutual enhancement of vulnerability to the other pathogen in patients with either disease and a consequent increase in the incidence and spread of AIDS. However, the mechanism of the process is not fully understood. The reduction of inhibitory capacity of our anti–HIV drug in vitro may reflect a common mechanism for production of the endogenous H/HS receptors for malaria and HIV-1, such as the specific expression of enzyme isoforms in the biosynthesis of anticoagulant H/HS. We will study this interpathogen effect on disease and antipathogen activity in an animal model.

Further preparation of Cp11 and CpC (smallest-mass component retaining high activity against cytopathological effects of HIV-1 in vitro) is ongoing. We will use CpC to prepare conjugates for immunological studies on SOLIS, e.g., to obtain a potent immunogen to produce an antibody for direct measurement of SOLIS concentration in the blood after intravenous administration. We plan to purify further CpC 8b regions to be used for advanced Fourier transform infrared spectral analysis and Heteronuclear Multiple Quantum Coherence correlation spectra to determine the relation of alternative chair structure to inhibition of HIV-1 gp120 and gp41.

Heparin-mimetic sulfated oligoxylan inhibitors of malaria parasites

Stone, Chen; in collaboration with Milhous, Sacci

Malaria is one of three deadliest diseases globally (more than 300 million cases annually). P. falciparum causes cerebral malaria and the death of about 2 million people annually (90 percent of whom are young children). There is no vaccine against malaria, which is becoming increasingly resistant to drugs. We are studying the antimalaria components of our S-OligoS (1) as in vitro inhibitors against primary invasion of hepatocytes and by (2) identifying S-oligoS component(s) against cerebral malaria in young children in a rosetting assay and (3) elucidating potential combined therapy for HIV infection and malaria (see above).

The initial infective process of malaria takes place in hepatocytes. The portal for invasion implicates the H/HS receptor systems. Heparin inhibits the rosetting of and cytoadhesion of parasitized P. falciparum erythrocytes (PfRBC) to normal RBC and the endothelium and clears blockage of the microcirculation, which ameliorates cerebral malaria in children. To explore the antimalarial potential of our heparin-mimetic S-oligoS, produced as anti–HIV drugs, we characterized its inhibition of malaria. We found that structural specificity and concentration governed the ability of S-OligoS to inhibit the invasion of hepatocytes by freshly isolated P. yoelii sporozoites; highest potency resided in two S-OligoS with mass of about 7,200 (Cp6) and 3,700 (Cp11). Cp11 has negligible antithrombin capacity, and we are preparing more of this S-oligoS. In contrast to sporozoite invasion of hepatocytes, components with mass less than 4,500 exhibited low inhibitory capacity toward the erythrocyte invasion stage of malaria while high potency was associated with a mass equal to or greater than 10,000, suggesting that inhibition of the two parasite stages differ. Preparation of additional Cp11 and expansion of our library in the mass range about 3,000–4,000 is under way, and we will continue to study inhibition of sporozoites, parasitized RBC, and rosetting as a method for stable, inexpensive, heparin-based antimalarials against infection, pathologies, and/or acute cerebral malaria.

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

Coxon

Our goal is to define the stereochemical dependence of nitrogen-15–proton coupling constants in amino sugars by high-resolution NMR measurements of such constants in model compounds. Our current experimental method of choice is the Carr-Purcell-Meiboom-Gill heteronuclear single quantum multiple bond correlation (CPMG-HSQMBC) NMR procedure. Using data we obtained for amino sugar derivatives during the past year, we expanded our project from defining the dihedral angular dependence of the coupling constants to include the general stereochemical dependence of these couplings, which also involves dependence on atomic electronegativities and their orientation. We plan to apply the nitrogen-15 NMR data to determine or verify the structures of nitrogen-containing bacterial polysaccharides and aminoglycoside antibiotics with examples from the fortimicin group.

We have completely analyzed the one-dimensional (1D) ¹H spectra and 1D ¹H–¹⁵N CPMG-HSQMBC NMR spectra of the subject amino sugars (with ¹⁵N at natural abundance) and correlated the resulting ³J_HCCN coupling constants with molecular geometry. Commonly present in bacterial polysaccharides, the amino sugars we are analyzing include d-glucosamine, d-mannosamine, and d-galactosamine; for this study we are using the sugars’ N-acetyl and hydrochloride derivatives. In a molecular modeling study using molecular dynamics (MD) calculations with energy minimization by molecular mechanics (MM) and implicit solvent, we defined the geometries of the alpha and beta pyranose anomers of N-acetyl-d-glucosamine, N-acetyl-d-mannosamine, and N-acetyl-d-galactosamine. The study yielded values of the vicinal nitrogen-hydrogen dihedral angles (φ) that we have correlated with measured values of the ³J_HCCN coupling constant.

Using MD/MM, we found that gauche arrangements of the hydrogen and nitrogen-15 nuclei in the common N-acetyl amino sugars had φ values in the range of 52 to 66, which corresponded to ³J_HCCN values of 0.8 to 2.5 Hz. Trans orientations of the hydrogen and nitrogen-15 nuclei displayed φ values in the range of 175 to 178 in MD/MM, which correlated with larger ³J_HCCN values in the range of 3.2 to 4.0 Hz. However, if the coupling involves the anomeric proton ¹H, then the magnitude of the trans coupling constant is reduced by the electronegativity of the pyranose ring oxygen to 2.7 to 2.8 Hz. Thus, it appears that the magnitude of the ³J_HCCN coupling constant could be used to distinguish gauche and trans orientations of vicinal nitrogen-15–proton pairs in amino saccharides, offering the possibility of defining the stereochemical configuration (axial or equatorial) of the nitrogen atom in unknown structures. Even though these common amino sugar models contain only limited ranges of ¹⁵N–¹H dihedral angles, the results for their ³J_HCCN coupling constants generally agree with those measured for the organic-soluble amino-sugar derivatives from which we previously formulated a Karplus-type equation: ³J_HCCN = 3.1 cos2 phi - 0.6 cos phi + 0.4.

Coxon B. A Karplus equation for ³J_HCCN in amino sugar derivatives. Carbohydr Res 2007;342:1044-1054.

NMR verification of structures of bacterial saccharide precursors for vaccines

Coxon, Schneerson, Kubler-Kielb, Mocca, Nelson, Szu

High-resolution NMR 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 function is to provide analytical NMR support as needed for current vaccine projects in the laboratory that involve the characterization of saccharide structure and purity. We investigate new vaccine-related substrates as they are developed.

Using 1D ¹H and 1D ¹³C NMR, we have analyzed about 40 monosaccharide and oligosaccharide intermediates for the preparation of a hexasaccharide-protein conjugate by high-resolution NMR at 500 MHz. When we needed additional information on spectral assignments, we used two-dimensional (2D) methods such as correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and heteronuclear single quantum correlation spectroscopy (HSQC). We based validation of the chemical structures on integration of the 1D ¹H NMR spectra and on the counting of carbon resonances in the ¹³C spectra, especially in well-separated spectral regions, such as those of the C = O, aromatic C, C-1, C-N, and C-CH₃ moieties. We also used 1D ¹³C distortionless enhancement by polarization transfer (DEPT) to differentiate ¹³CH₂ resonances from those of ¹³CH and ¹³CH₃. This simple spectral editing technique yields a ¹³CH₂ count in the form of ¹³C resonances of inverted phase and was particularly applicable to these intermediates because of the mixed CH, CH₂, and CH₃ resonances of the amide and linker side-chains in the structures.

We provided NMR service spectra measured at 500 MHz for the identification of 20 polysaccharide fragments isolated from Shigella sonnei, including products of nitrous acid deamination that are used in structural studies and spectra that facilitate the detection of core polysaccharide in the samples. The techniques include 1D ¹H and ¹³C NMR but sometimes 2D COSY, TOCSY, and HSQC as well. We acquired the spectra at an elevated sample temperature of 50°C to reduce the viscosity of the solutions, thus permitting the measurement of sharper spectra. The techniques provide complex fingerprint spectra that lend themselves to comparison with data in the literature in order to verify identity. We also measured service spectra for nine fractions isolated from Klebsiella pneumoniae type 2, including a series of three samples of the polysaccharide that had been subjected to sonication for 5, 15, or 30 minutes to test a molecular weight–reduction procedure. We identified one sample of a polysaccharide found in S. flexneri type 2a by 1D ¹H and ¹³C NMR.

Isolation and NMR elucidation of three saponins from Blighia sapida

Coxon; in collaboration with Mazzola, Kennelly, Parkinson, Freedberg

Saponins are steroid glycosides, steroid alkaloids (steroids with a nitrogen function), or triterpenes that are found especially in plant skins, where they form a waxy, protective coating. They may be useful in the human diet for controlling cholesterol, but some are poisonous or may cause urticaria. We set out to isolate three saponins from Blighia sapida and to determine their structures by high-resolution NMR spectroscopy and mass spectrometry.

QS-21 is a complex saponin that has been used as a vaccine adjuvant, thereby stimulating interest in this general class of compounds. In a collaborative study, we isolated three complex saponins—Blighosides A, B, and C—from the tree Blighia sapida, determined their structures by high-resolution NMR, and obtained their molecular weights by mass spectrometry. Our results show that Blighoside A (MW 1086 Daltons) has an unbranched tetrasaccharide containing arabinose, rhamnose, glucose, and a second arabinose linked through one of the arabinose moieties to C-3 of a 30-carbon triterpene aglycone hederagenin. The rhamnose is linked to C-2 of the first arabinose, the glucose to C-3 of the rhamnose, and the second arabinose to C-4 of the glucose, which is acetylated at O-3. Blighoside B (MW 1,070 Daltons) contains the same tetrasaccharide moiety as Blighoside A but is linked to a slightly different triterpene, oleanolic acid, in which C-23 of hederagenin is a methyl group instead of a hydroxymethyl group. Blighoside C (MW 1,504 Daltons) has six monosaccharide units linked to oleanolic acid. Attached to C-3 of this triterpene is a linear hexasaccharide consisting of two xyloses, rhamnose, glucose, a second rhamnose, and another glucose. The second xylose is linked to C-3 of the first xylose, the first rhamnose to C-2 of the second xylose, the first glucose to C-3 of the preceding rhamnose, the second rhamnose to C-4 of the preceding glucose, and the final glucose to C-3 of the preceding rhamnose. In addition, the first glucose is acetylated at O-3 and O-6 and the terminal glucose at O-4 and O-6.

The NMR methods used for determining structure included 1D slices of 2D TOCSY data sets to generate separate sub-spectra for the individual monosaccharide residues, which allowed identification of sugar type by measurement of vicinal ¹H–¹H coupling constants. We also used 2D HMBC experiments that gave ¹H–¹³C through-bond connectivities that defined nuclear neighbors in the structures as well as the sugar linkages and positions of acetyl substitution. 2D ROESY (rotating-frame NOE spectroscopy) experiments indicated ¹H–¹H through-space interactions, especially for proximal protons in adjacent sugar residues. We achieved additional resolution of the NMR signals of similar sugar residues with 2D ¹H-coupled HSQC spectra, which also allowed identification of equatorial or axial protons in the sugars and determination of their anomeric configuration by measurement of ¹J_C-1,H-1 coupling constants.

NMR analysis of synthetic oligosaccharide fragments of the OSP of E. coli O-148

Coxon, Pozsgay

In efforts related to preparation of a potential vaccine against Shigella dysenteriae type 1, we acquired 1D and 2D high-resolution NMR spectra at 500 MHz from synthetic oligosaccharide fragments of the O-SP of E. coli O-148, which cross-reacts with S. dysenteriae type 1. The repeating units of the O-SPs are closely related; the main difference is the presence of d-glucose units in the E. coli OSP instead of d-galactose in S. dysenteriae type 1. Studies of a tetrasaccharide (4-mer), octasaccharide (8-mer), and dodecasaccharide (12-mer) are under way, including one, two, or three repeating units of the O-SP of E. coli O148. We derivatized these oligosaccharides as their 5-methoxycarbonylpentyl glycosides in advance of coupling to a carrier protein that would be used to prepare a protein-saccharide conjugate.

We employed several NMR techniques, including 1D ¹H, 1D ¹³C, 2D ¹H–¹H COSY, 2D ¹H–¹H TOCSY, 2D ¹³C–¹H HSQC, 2D ¹³C–¹H heteronuclear multiple bond correlation (HMBC), 2D ¹H-coupled ¹³C–¹H HSQC, and 1D ¹H-coupled ¹³C NMR. COSY yields information about ¹H spectral assignments, TOCSY provides separate ¹H sub-spectra for individual sugar residues, HSQC provides ¹³C spectral assignments by correlating the ¹H and ¹³C chemical shifts, and HMBC pulse sequence delineates the positions of substituents by correlating their ¹H or ¹³C chemical shifts with those of nuclei in the sugar rings. The ¹H-coupled 1D ¹³C and ¹H-coupled 2D HSQC techniques measure ¹³C–¹H coupling constants of the oligosaccharides. The latter method is particularly useful when the ¹H-coupled, 1D ¹³C NMR spectra are insufficiently dispersed for a facile analysis. We measured ¹J_C-1,H-1 values, which typically yielded information on the configuration (alpha or beta) of all the anomeric linkages in the oligosaccharide. Confirmatory information is often available from the ³J_H-1,H-2 values measured from the ¹H NMR spectra, with the exception of mannopyranose and rhamnopyranose, for which the values of the alpha and beta anomers are similar.

We encountered a problem with broadness of the ¹H spectra of the 8-mer and 12-mer in D2O solutions, which could inhibit the analysis and assignment of the spectra. Surmising that such broadness might be attributable to the viscosity of the solutions of the 8-mer and 12-mer, we acquired ¹H and ¹³C spectra of the 12-mer at elevated temperatures of 50°C and 60°C but discovered that the higher temperatures did not resolve the issue. We are investigating the possibility that the unexpected broadness of the spectra results from paramagnetic metal ion contaminants in the 8-mer and 12-mer. Carbohydrates are often excellent metal-complexing agents, and the occasional spectral broadening observed in the presence of scavenged paramagnetic metal ions is a well-known phenomenon. In an effort to remove any polyvalent, paramagnetic ions, we have therefore treated the 8-mer with Chelex 100 cation exchange resin (sodium form). Following treatment, the 8-mer showed improved ¹H spectral resolution.

Collaborators

Amina Ahmed, MD, Carolina Medical Center, Charlotte, NC
Shai Ashkenazi, MD, Schneider Children’s Hospital, Tel Hashomer, Israel
Parvin Azimi, MD, Children’s Hospital and Research Center at Oakland, Oakland, CA
Joseph Bellanti, MD, Immunology Center, Georgetown University Medical Center, Washington, DC
Dianjun Cao, PhD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
George Carlone, PhD, Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, GA
Yehuda Chowers, MD, Sackler School of Medicine, Tel Aviv University, Tel Hashomer, Israel
John Clements, PhD, Tulane University School of Medicine, New Orleans, LA
Jerri Curtis, MD, Laboratory of Biochemistry, NHLBI, Bethesda, MD
Daron I. Freedberg, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Magnús Gottfredsson, MD, University of Iceland School of Medicine, Reykjavik, Iceland
Patricia Griffin, MD, Centers for Disease Control and Prevention, Atlanta, GA
Tyra Grove Krause, MD, PhD, Statens Serum Institut, Copenhagen, Denmark
McDonald K. Horne, III, MD, Hematology Service, Clinical Center, NIH, Bethesda, MD
Yasutaka Hoshino, DVM, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Michael F. Howitz, PhD, Statens Serum Institut, Copenhagen, Denmark
Albert Z. Kapikian, MD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Edward J. Kennelly, PhD, Lehman College, City University of New York, West Bronx, NY
Dennis J. Kopecko, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Teresa Lagergard, PhD, Göteborg University, Göteborg, Sweden
Stephen H. Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
Rodney L. Levine, MD, PhD, Biochemistry and Biophysics Center, NHLBI, Bethesda, MD
Marc S. Lewis, PhD, Intramural Research Program, NIBIB, Bethesda, MD
Maria O. Longas, PhD, Purdue University, Hammond, IN
Eugene P. Mazzola, PhD, University of Maryland and University of MD-FDA Joint Institute, College Park, MD
James McMahon, PhD, Molecular Targets Development Program, NCI, Frederick, MD
Will Milhous, MD, Walter Reed Army Institute of Research, Silver Spring, MD
Louis Miller, MD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
Mark A. Miller, MD, Fogarty International Center, NIH, Bethesda, MD
Kåre Mølbak, MD, Statens Serum Institut, Copenhagen, Denmark
Sophie E. Moore, PhD, London School of Hygiene and Tropical Medicine, London, UK
Patrick R. Murray, PhD, Department of Laboratory Medicine, Clinical Center, NIH, Bethesda, MD
Michael Osterholm, PhD, MPH, Center for Infectious Disease Research and Policy, University of Minnesota, Minneapolis, MN
Ainsley Parkinson, BS, Lehman College, City University of New York, West Bronx, NY, and University of MD-FDA Joint Institute, College Park, MD
Donald C. Robertson, PhD, College of Veterinary Medicine, Iowa State University, Ames, IA
Sandra Romero-Steiner, PhD, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA
John Sacci, Jr., PhD, University of Maryland, Baltimore, MD, and Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD
Joseph Shiloach, PhD, Biotechnology Core Laboratory, NIDDK, Bethesda, MD
Darrell Singer, MD, MPH, Walter Reed Army Institute of Research, Rockville, MD
James F. Troendle, PhD, Biometry and Mathematical Statistics Branch, NICHD, Bethesda, MD
Evgeny Vinogradov, PhD, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
Leonard E. Weisman, MD, Baylor College of Medicine, Houston, TX
Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact robbinsjo@mail.nih.gov or robbinsjo@mail.nih.gov.