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Home > Section on Bacterial Disease Pathogenesis and Immunity

Development of Vaccines Against Bacterial Diseases Especially in Children

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

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

Following the widespread use of Haemophilus influenzae (Hi) type b (Hib) conjugate vaccines and the consequent near-elimination of Hib, Hia is the most prevalent Hi type causing invasive disease, especially common among certain children populations. Our goal is to provide a vaccine against Hia, A second goal is to investigate the feasibility of providing a single vaccine to several pathogens. Hib and Hia are the most virulent Hi types and exhibit resistance to the bactericidal effects of complement alone. Both are composed of a neutral sugar (ribose or glucose), an alcohol (ribitol), and a phosphodiester. The structural, experimental, and clinical properties of Hia CP closely resemble those of Hib, and the increasing number of reports of Hia-invasive disease suggests that development of a Hia conjugate is warranted.

The properties of Hia and Hib CPs are similar and methods for conjugating type b CP to a protein are applicable to Hia. D-1,5-ribitolphosphate is a constituent of both CPs. 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 its conjugate cross-reacted with both Hia and Hib. We synthesized polyribitolphosphate chains containing 8 or 12 repeat units, with the terminal keto groups used for conjugation to aminooxylated bovine serum albumin (BSA) or to tetanus toxoid. The conjugates were injected into mice, 3 times, 2 week apart, 2.5ug/mouse. Antibodies to both Hia and Hib were measured by ELISA, the octamer conjugate being a better immunogen than the dodecamer conjugate. Some of the tested sera showed bactericidal activity against both Hia and Hib, roughly correlated with their ELISA values. Other constructs and formulations are being studied. Clinical lots are expected to be studied for safety and immunogenicity in adults and children. There are too few Hia cases 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, meningococcal groups Y and W135 and poliovirus type 2, was based on their safety and immunogenicity.

Bordetellae spp.

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 thereby 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, Gram-negative bacilli, cause respiratory tract infections in mammals and in birds. B. pertussis, B. parapertussis, and B. bronchiseptica are 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. Based on 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 from B. bronchiseptica and B. parapertussis 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 later two. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SPs. We prepared conjugates of B. bronchiseptica and B. parapertussis O-SP using either the Kdo residue exposed by mild acid hydrolysis of the LPS or the core glucosamine residue exposed by deamination of the LPS. Both coupling methods were done at a neutral pH, at room temperature, and in a short time. The conjugates were 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 because of the absence of a 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 structures. B. pertussis expresses only the core region saccharide (OS), composed of 12 sugars. B. bronchiseptica, easier to cultivate 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) Heptose is phosphorylated 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 measured by ELISA. Conjugates were also prepared of oligosaccharides of B. bronchiseptica mutants, and their immunogenicity is being evaluated.

Shigellae

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, fold increases in antibody levels were similar to those for adults, but the actual achieved levels were lower.

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 BSA, or to non-toxic recombinants of diphtheria toxin or C. difficile toxin B. The coupling reaction was performed at a neutral pH, at room temperature, and in a short time. Levels of IgG anti–S. sonnei LPS 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 S. sonnei core structure of 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 these studies the structure to the P. shigelloides O-SP, has been assigned.

Vaccine development for Group B Neisseria meningitidis and Escherichia coli K1

 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 polymer that is identical to the E. coli K1 capsular polysaccharide (CP) of as well as to host structures, especially during fetal development. 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 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.

Of the 13 known meningococcal CP groups, 5 (A, B, C, W135, and Y) cause almost all meningococcal disease. As essential virulence factors, the five group CPs inhibit the protective actions of complement and function as protective antigens; a critical level of specific serum IgG anti CP induces both complement-mediated lysis of groups A, C, W135, and Y and opsonophagocytic killing of group B. Effective CP-based vaccines exist for groups A, C, W135, and Y, but none for GBM or E. coli K1. GBM causes more infections in infants and young children than do groups A, C, W135, or Y.

Although PSA antibodies bind to fetal and adult tissues in vitro, there is no evidence for in vivo binding or associated pathology. Efforts have been directed towards developing vaccines using 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, subject to antigenic variation, and may not be representative of all GBM strains. 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) conjugate vaccines, we developed a PSA conjugate that induced protective levels of serum IgG anti–PSA. It is simple to produce, easy to standardize and should be close to 100 percent effective at all ages. Its performance in laboratory mice and primates has been confirmed.

Few studies have compared severity of infection and outcome among meningococcal serogroups, and published studies of sequelae of meningococcal meningitis do not mention autoimmune diseases, such as Guillain-Barré syndrome, multiple sclerosis, etc. 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 GCM patients or people 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 the offspring for up to 31 years after meningococcal disease.

Peptide-protein conjugate vaccines

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. A recombinant PA from an uncapsulated strain, formulated with formaldehyde and/or alum was immunogenic in mice. These formulations were safe in adult volunteers. Local and systemic reactions were rare and minor and preliminary antibody assays compare favorably with those induced by the licensed vaccine.

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 the higher immunizing dosages. The use of alum increased PA antibody levels with little effect on anti–PGA levels.

Chimpanzees were immunized, 10 mcg PGA/animal, sc; one with rPA-PGA another with TT-PGA, with the goal of preparing humanized monoclonal antibodies to PGA. Both chimps responded with antibodies to both vaccine components. Higher anti PGA levels were obtained in the TT-PGA injected chimp. PGA specific IgG1 and IgG3 were prepared.

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 flagellae of Pseudomonas syringae, 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, measured by ELISA, and to B. anthracis spores, detected by fluorescence microscopy. We propose the use of Shewanella CP conjugates as a component of an anthrax vaccine.Analysis of Shewanella MR-4 LPS carbohydrate backbone showed 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. The goal of this study is to provide 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 to the asexual, human stage of the parasite; CSP was cloned into E. coli, isolated and several formulations, including adsorption onto alum, were evaluated in young outbred mice. Based on our studies with peptides of the B. anthracis capsule, peptides of 4-5 repeat units of NANP were synthesized and bound 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 the sporozoites in IFA. NANP-Pfs25 conjugates induced long lasting antibodies to both vaccine components; 3 months serum levels were higher than those taken 1 week after the last injection. Alum adsorbed CSP induced higher antibody levels than the non-adsorbed protein. The terminal amino acid of the CSP-derived peptides was shown to be an important determinant, with NPNA-protein being the best immunogen.

The second approach was directed to the sexual, mosquito parasite stage, to provide a transmission blocking vaccine. Pfs25, a low molecular mass protein, non immunogenic by itself, was bound onto 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 3 and 7 months after immunization were higher than 1 week after the last injection. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased further the antibody levels. Transmission blocking activity of immune sera correlated with antibody levels measured by ELISA.

Protein and polysaccharide conjugate vaccines to enteric diseases

 The capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine with limited efficacy in children less than 5 years old. To provide a vaccine for younger children, Vi was conjugated to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). An efficacy of 89% at 47 months was shown in 2-to-5-year olds injected with Vi-rEPA. Vi-rEPA was administered to 301 Vietnamese infants concurrently with their routine immunizations at 2, 4, 6 and 12 months. Controls received Hib-TT +DTP or DTP. No serious adverse events occurred in any group. The post immunization GM IgG anti-Vi level in the Vi-rEPA group was significantly higher than in the control groups. There was no difference in the level of IgG anti-DT among all groups. Antibody levels to TT and PT are being assayed.

Fruit pectin is composed of polygalacturonic acid, structurally identical to the backbone of Vi. Pectin was O-acetylated chemically at the C2 and C3 positions (OacPec) to mimic Vi antigenically. A phase I study of OacPec-rEPA injected once into 25 healthy adult volunteers showed it to be safe.  Six weeks after injection, a  > 4 fold rise in IgG anti-Vi was found in 60% of the volunteers. No rise in IgG anti-pectin was found.

Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries transmited through ingestion of food or drink contaminated by infected persons. The presence of chronic carriers of SPA is not established. In collaboration with the Guangxi Center for Disease Control and Prevention, we investigated a large outbreak in a residential middle school. A chronic carrier, most likely the source of the outbreak, was identified by an exceptionally high level of serum IgG anti-LPS and a positive rectal swab. An expanded survey, using the same methods, was initiated.

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 its LPS. In a phase 1 trial, O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. To enhance immunogenicity, a conjugation scheme was devised employing derivatization of both the carrier protein and the O-SP with heterobifunctional reagents. The improved O-SP conjugates and conjugates of a V. cholerae O-SP hexamer we synthetized induced higher vibriocidal activities than the conjugate used in the Phase I study.

Rotavirus is the most common cause of infantile diarrhea worldwide. Two rotavirus vaccines are licensed, a live attenuated and a reassortant, both orally administered. We are designing a parenteral vaccine based on capsid proteins. Recombinant capsid proteins with truncated C or N termini were expressed in E. coli. Mice injected with the recombinant proteins developed neutralizing antibodies. Conjugation of the recombinant proteins to polysaccharide increased their solubility

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.

Synthetic vaccines against human pathogenic bacteria

 Protein conjugates of oligosaccharide (OS) fragments of bacterial polysaccharides (PS), such as capsular PS and lipopolysaccharides, may elicit antibodies and be protective against the homologous organism. Our work targets vaccines against Shigella dysenteriae type 1, E. coli O148, Borrelia burgdorferi, and Haemophilus influenzae type a.

The O-specific PS (O-SP) of S. dysenteriae type 1, causing bacterial dysentery, is a prime target for vaccine development. It contains L-Rha, D-Gal, and GlcNAc residues. We found that conjugates of various proteins with fragments of the O-SP in the 8-16-mer range elicit O-SP-specific Ab in mice. The highest levels were obtained with conjugates of 12 and 16-mer containing an average of ten saccharide chains per protein. Higher anti-O-SP levels were achieved when the non-reducing terminus was occupied either by a D-Gal or by a D-GlcNAc instead of an L-Rha residue. We have synthesized cllnical lots of the 10- and 11-mers equipped with a linker for conjugation to proteins.

The O-SP of E. coli O148, a human enteropathogen, differs from that of S. dysenteriae type 1 in that the D-Gal residue in the latter is replaced by a D-Glc. We have prepared BSA-conjugates of synthetic tetra-, octa-, and dodeca-saccharide fragments of E. coli O148 by single-point attachment and used the conjugates to elicit Ab in mice. These Ab cross-react with the O-SP of S. dysenteriae type 1. This finding raises the possibility that a vaccine against S. dysenteriae type 1 might protect against both organisms.

Two major glycolipids (BBGL-1 and -2) secreted by Borrelia burgdorferi, the causative organism of Lyme disease, have been synthesized in their native form and one that can be conjugated. These are being studied for their immunogenicity as a prelude to developing a conjugate vaccine against this disease.

We are developing a conjugate vaccine against H. influenzae type a by preparing chemically fragments of its capsular material which is constructed of glucosyl-ribitol-phosphate repeating units. We have synthesized the glucosyl-ribitol unit and are preparing the complete repeating unit to use for chain extension, conjugation to proteins, and evaluation of conjugate immunogenicity.

The structures of all new compounds were verified by detailed NMR analyses.

Neonatal respiratory distress related to colonization with group B streptococci

Clinical features of early-onset GBS disease are similar to those of endotoxic shock, and respiratory distress is a prominent symptom. Respiratory distress may occur in newborns, without a known GBS infection. Phospholipids from group B streptococcal (GBS) cell wall cause pulmonary hypertension in experimental animals. When exposed to penicillin, Streptococcus mutans releases phospholipids immediately. We hypothesize that GBS phospholipids (cardiolipin) may lead to pulmonary hypertension and respiratory distress in GBS colonized newborns, especially those of penicillin treated mothers. The goal and objective of this study were to examine clinical and epidemiologic data related to this potential association. 

A prospective study aimed at relating serum GBS cardiolipin levels to respiratory distress in newborns of GBS colonized mothers was conducted in two U.S. academic centers Baylor College of Medicine, Houston, Texas and Children’s Hospital and Research Center Oakland, California. From 2/5/08 to 2/4/09, vaginal and rectal swabs were obtained during labor from 5,562 mothers of ≥32 weeks gestation for GBS culture; 1,032 (18.6%) were colonized.  A semiquantitative culture was used to determine the degree of colonization. Demographic and clinical data were obtained by the review of medical records. Data analyses are underway. Three groups of GBS colonized mothers are being assembled: untreated, -lactam antibiotic-treated, and treated with non- -lactam antibiotics. Maternal and cord blood samples were collected for the cardiolipin assay. All newborns are monitored for signs of respiratory distress. Two groups of newborns are being assembled: with and without respiratory distress.

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

Vaccination is the most effective method of controlling infectious diseases; however, a rapid influenza vaccine response is impossible because outdated manufacturing methods. In our approach to this challenge, we developed a protocol for rapid production of recombinant hemagglutinin protein (rHA) for use as a vaccine against rapidly spread pandemic influenza viruses. The HA gene was optimized for expression in E. coli and synthesized de novo without need of access to pathogenic virus strains.  Rapid (rPCR) protocols also have been established for construction of HA genes.  Our response vaccine time is reduces to less than one month compared to the 6-8 month traditional egg-based technology. We have standardized downstream processing for protein extraction, purification, refolding, and vaccine formulation and have produced pilot plant quantities of recombinant HA from five representative influenza A viruses including H1N1 2009 swine flu pandemic. Formulated vaccines of rHA protein are currently undergoing immunization and protection evaluations. Preliminary studies indicate that alum-absorbed HA induced antibodies with hemagglutination inhibition titers of 40 or higher, suggesting that rapidly produced rHA could induce protective immune responses against influenza virus infection. Recent literature reports suggest that antibodies to the exposed N-terminal 23 amino acids (M2e) of the highly conserved mature matrix 2 protein (M2) of influenza viruses may ameliorate disease symptoms.  We conjugated chemically a synthetic M2e peptide to a genetically detoxified diphtheria toxin carrier protein.  This alum adsorbed conjugate induced high IgG anti-M2e. 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

The heparin glycobiochemistry lab studies molecular processes by which multi-functional heparin/heparan sulfate modulator biopolymers (H/HS) govern growth in children and lifetime well-being. Knowledge of the roles of H/HS in normal and disease processes, will lead to development of drugs to prevent or treat diseases, such as HIV-1 and malaria.

The first aim is developing a combinatorial library of heparin-mimetic drugs (oligosaccharides, S-OligoS) to establish structure-function relations and identify those components that act against pathogens and their sequelae, and might help to identify protective antigens. Second is to develop a novel anti-AIDS drug (SOLIS) from a component (Pk II, CpF) which we identified as a potent in vitro inhibitor of HIV-1. Third is to determine the compatibilities and inhibitory effects of SOLIS and Cp11, a potential anti-malaria drug, in a combined formulation for HIV infection in malaria endemic regions. Physical, chemical and optical techniques are applied, modified and/or devised to determine primary structures and conformations of the S-oligoS to elucidate their functional heparin-mimetic sulfate and carboxyl geometries. Equipment and biological regents are devised and prepared for automation and as clinical markers.

Summary

HIV-infection in Americans is 50 times that worldwide and rising. There is neither cure nor a fully effective vaccine, long term control of viral load is compromised by drug toxicity and multi-drug resistant strains, and protease and reverse transcriptase inhibitors are the principal treatments. Inhibitors against viral components having other functions in HIV-1 attack are critically needed. Heparin was shown to inhibit binding and fusion of AIDS virus to its target CD4 cell in vitro, but pharmaceutical heparin is an antithrombin-activator anticoagulant and may not be useful clinically because of possible hemorrhagic toxicity.

We discovered that heparin assumed a helical secondary structure in solution. We classified the various sulfated glycosaminoglycans (GAG, mucopolysaccharides) based on their optically active structures, and demonstrated that H/HS (and keratosulfate) comprise a separate class of GAGs. H/HS are highly sulfated, found in cells and matrix and comprise modulator/receptor systems that govern the level of activity/function of normal proteins and cell systems, but also serve as receptors for human pathogens. H/HS have a large diversity in sugar modifications and sulfate density on their polysaccharide chain. These unique sequences can account for its modulation of diverse physiological processes. e.g., development, growth, inflammation, anticoagulation, and susceptibility to human pathogens. Due to the similarity of properties of H/HS chain sequences, libraries of unique H/HS have not been obtainable for research. We developed a combinatorial heparin-mimetic family (library) based on the structure-function model of antithrombin-activating heparin of Rosenberg, et al. from a German pharmaceutical, chemically sulfated xylan. It is comprised of S-oligoS that mimic heparin in most of its biological actions, at similar concentrations. With this we demonstrated for the first time that in vitro inhibitions of HIV-1 cytopathology and syncytium-formation were 1) governed by structural specificity and 2) each separable from the anticoagulant S-oligoS of the pharmaceutical and from each other, two essential indicators of their usefulness for further drug development. We devised procedures for clinical preparation of a highly active HIV-1 virus fusion inhibitor that is free of anti-thrombin toxicity (PK II, SOLIS).

Summary of properties of PK II:

  1. IC 50 vs cell-killing (formazan assay) at 0.20-0.3 mcg/ml
  2. IC 50 vs virus-cell fusion at 0.05-0.1 mcg/ml
  3. Inhibition of adherence of CD4 cells to gp120 (concentration-dependent)
  4. Anticoagulation vs thrombin activity at less than 0.05-0.3 HU/mg
  5. Endotoxin content less than 0.06 eu.

Solis, a potential Hiv-1 viral entry inhibitor

We are completing a clinical preparation of SOLIS for formulation in a Phase I, iv-administration trial. SOLIS would be an adjunct inhibitor of HIV-1 entry into CD4 cells. Heparin is known to affect conformation of proteins thereby modulating their biological function. Inhibition of conformational changes in gp 41, associated with formation of the fusigenic structure required for fusion progression, is a possible mechanism of action of SOLIS. An alternate receptor-driven mechanism not associated with the cell membrane is possible. Orally administered S-oligoS are poorly absorbed, indicating use of iv administration with a consequent need for a large dos. Clinical studies could be flawed by use of an indirect measure of blood levels of the drug. We produced appx.1.6 g of stage II SOLIS from a first lot of pharmaceutical starting material. Negotiations to renew our confidentiality agreement and a materials transfer with the pharmaceutical company were recently completed, with a donation of 40 grams for preparation of additional SOLIS.

Studies on combination therapy

Enlargement of the library by further fractionation of Cp11, CpC (purified from Cp7, 8A region), and Cp8B are in progress. CpC, the smallest component retaining high activity against HIV-1 cytopathology in vitro, will be used in structural studies and to prepare conjugates and produce an antibody reagent for direct measurement of blood SOLIS concentration after iv administration.

Virus ligands and bifunctional solis probe

CpC will be used to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe. Chemical bonding to viral/cell protein structures during virus attack will be used to isolate the putative endogenous protein-probe. Such ligand(s) could be used to obtain receptors which might be protective antigens.

Derivatization of S-Oligos

Derivatization will follow our procedures for mono-derivatizing S-oligoS at the reducing end. Synthetic polypeptides will be selected based on the mode of action of SOLIS for conjugation to produce immunologic reagents and immunogens.

Structure-function relations

We discovered that S-oligoS remaining after chemical sulfation of native xylan comprised a family of oligomers that range from about 20 to less than 2 KD. Compositional and structural analysis of Cps led us to propose a tetraS motif within the S-OligoS chains consisting of D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3. We also found axial sulfates (alternative chair conformations) in these S-OligoS are displayed in decreasing proportions with decreasing mass. Alternative chair conformation was unequivocally determined in SOLIS by HMQC correlation NMR analysis. Current preparations of Cp II, Cp 8b, and CPC will be used for advanced FTIR spectral analysis and HMQC correlation spectra to determine the relation between alternative chair structure in S-OligoS and inhibition of HIV-1 gp120 and gp41, and anticoagulation.

Heparin-mimetic sulfated oligoxylan inhibitors of malaria parasites

The main goal of this project is to develop heparin-mimetic drugs that prevent or treat diseases caused by pathogens known to be inhibited by heparins. One goal is to expand and apply our family of H/HS mimetic sulfated oligoxylans (S-OligoS) to identify potential anti-malaria parasite components. This includes further elucidation of a novel anti-malaria S-OligoS which we found to be a potent inhibitor of primary invasion of hepatocytes by parasites in vitro (Cp11). Our objectives are to improve preparative methods of specific S-OligoS inhibitors to elucidate their structure-function relation and usefulness in vaccine design. A second goal is to test our H/HS family in vitro in a rosetting assay, to identify potential components as agents against cerebral malaria in young children, for which there is no cure. A third goal addresses the predicted increase in incidence and spread of HIV infection for malaria endemic regions, due to a mutual increase in vulnerability of either patient to the other pathogen. Studies aim to elucidate the effects and caveats of co-dosing of the two S-OligoS to protect against the AIDS virus in malaria endemic areas.

The initial infective process of malaria in humans is invasion of hepatocytes by the parasite. In several studies, this invasion has implicated the heparin/heparan sulfate (H/HS) receptor systems. We discovered that heparin assumed helical structures in solution. We classified the sulfated glycosaminoglycan (GAG) polymers based on their optically active structures and demonstrated that H/HS (and keratosulfate) are multi-functional. Heparin also inhibits the rosetting and cytoadhesion of Plasmodium falciparum parasitized erythrocytes to normal RBC and the endothelium, which clears blockage of the microcirculation ameliorating life-threatening symptoms of cerebral malaria in children. H/HS are modulatory receptors, found in most tissues of the body where they govern the activity/function of physiological systems, and are receptors for pathogens. We have applied our library of heparin-mimetic S-oligoS to characterize the inhibition of malaria parasites in vitro and explore its usefulness as a treatment. We found that the heparin-based inhibition of hepatocyte invasion by freshly isolated P. yoelii sporozoites varied among library components and was concentration dependent. This indicates that inhibition is governed by structure. The highest potency was in two S-OligoS: Cp6 of 7200 D with 43 % inhibition at 3.5 mM and C11 of 3700D with 56 % inhibition at 5 mM. S-OligoS of 3700D have shown negligible antithrombin activity. Thus, Cp11 was selected for an enlarged preparation. Importantly, last year's efforts to renew a mutual confidentiality agreement with the German pharmaceutical company supplying the starting pharmaceutical material were successful and donation of 40 gm was recently received enabling the large preparation. Compositional and structural studies will be extended: 1) The purified Cp11 will be examined for the presence of a tetrasaccharide structure of three xyloses and a glucuronic acid as a branch on the xylan chain having high anionic charge density ((90 percent sulfated) which was exhibited by other H/HS mimetic S-OligoS; 2) FTIR spectroscopy revealed the presence of axial sulfates (alternative sugar ring conformation), which increased with mass in ratio of sulfates, and was demonstrated in the SOLIS structure by NMR studies. Such alternate forms induce distance and angle alterations in the otherwise uniform S-OligoS chain. Such a motif could accommodate the subtle variations in sulfate geometry that are required for multifunctional mimicry by heparins. Elucidation of the Cp11 structure might enable high throughput methods in further drug development. We previously suggested that co-treatment of malaria and HIV-AIDS would be feasible because of the close similarity in the physicochemical properties of the respective S-OligoS inhibitors. The recent report showing an increase in the rate of infection and spread of AIDS in malaria endemic areas prompted a preliminary study on the co-dosing of Cp11 (relatively inactive vs HIV-1) and SOLIS (malaria parasite and HIV-1 inhibitors) in vitro. Increasing ratios of Cp11 to SOLIS displayed a trend towards decrease in SOLIS capacity to protect against fusion between the virus and the target CD4 human line. Additional studies will be conducted.

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

A study of the Karplus-type angular dependence of 1H-15N NMR coupling constants of bacterially-related aminosaccharides and also aminoglycoside antibiotics is underway. A major review has been written on the application of the Karplus equation to the NMR coupling constants of carbohydrates, which is one of the key methods for determining the molecular geometry of saccharides.

NMR verification of structures of bacterial saccharide precursors for vaccines

 In relation to ongoing studies of Cholera vibrio antigens, a further 20 monosaccharide and oligosaccharide intermediates for the preparation of a hexasaccharide-protein conjugate have been analyzed for structure and purity by high-resolution one-dimensional (1D) 1H and 1D 13C NMR spectroscopy. Validation of the chemical structures was based on integration of the 1D 1H NMR spectra, and on counting of 13C resonances.

Various polysaccharide fractions prepared from wild and mutant strains of Shigella flexneri type 2a have been characterized by 1D 1H NMR, particularly with respect to assessing the content of core saccharide by integration of the polysaccharide H-1 and rhamnose methyl signals.

A new preparation of the capsular polysaccharide of Haemophilus influenzae type a has been investigated by 1D 1H and 13C NMR and found to have excellent purity, and close agreement with the spectra of previous preparations in this laboratory.

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

 Detailed assignments have been obtained for the 1D and 2D high-resolution NMR spectra of synthetic tetra-, octa-, and dodeca-saccharide fragments of the O-specific polysaccharide (OSP) of Escherichia coli O-148, which cross-reacts with Shigella dysenteriae type 1 as a consequence of the close structural similarity of the OSPs of these pathogenic bacteria.

The structures of two synthetic galactosyl diglyceride antigens for Borrelia burgdorferi (Lyme disease) have been proved by high-resolution 1D and 2D NMR, using complete assignments of their 1H and 13C NMR spectra. Several correlations of the 13C chemical shifts of carbonyl carbon nuclei with substituent type and position have been developed.

Publications

  • Robbins JB, Kubler-Kielb J, Vinogradov E, Mocca C, Pozsgay V, Shiloach J, Schneerson R. Syntheses, characterization and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. PNAS 2009 106:7974-7978.
  • Biesova Z, Miller MA, Schneerson R, Shiloach J, Green KY, Robbins JB, Keith JM. Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus. Vaccine 2009 epub:ahead of print.
  • Howitz MF, Simonsen J, Krause TG, Robbins JB, Schneerson R, Mølbak K, Miller MA. Risk of adverse birth outcome after group B meningococcal disease: results from Danish National Cohort. Pediatr Infect Dis J 2008 28:199-203.
  • Robbins JB, Schneerson R, Keith JM, Kubler-Kielb J, Miller MA, Trollfors B. Pertussis vaccine. A critique. Pediatric Nfect. Dis. J. 2009 28:237-241.
  • 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 physicochemical and immunological characterization of the conjugates. Vaccine 2008 26:3587-3593.
  • Coxon, B. Developments in the Karplus equation as they relate to the NMR coupling constants of carbohydrates. Advances in Carbohydrate Chemistry and Biochemistry 2009 62:17-82.

Collaborators

  • 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
  • John Clements, PhD, Tulane University School of Medicine, New Orleans, LA
  • Jerri Curtis, MD, Laboratory of Biochemistry, NHLBI, 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
  • Teresa Lagergard, PhD, Göteborg University, Göteborg, Sweden
  • Stephen H. Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
  • Rodney L. Levine, 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
  • James McMahon, PhD, Molecular Targets Development Program, NCI, Frederick, 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
  • Sandra Romero-Steiner, PhD, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA
  • Joseph Shiloach, PhD, Biotechnology Core Laboratory, NIDDK, Bethesda, 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
  • Kim Y. Green, PhD, Epidemiology Service, NIAID
  • Richard J. Webby, PhD, Saint Jude Children’s Research Hospital, Memphis, TN
  • Peter Backlund, PhD, Section on Mass Spectrometry and Metabolism, NICHD
  • Yimin Wu, PhD, Lab of Malaria Immunology and Vaccinology, NIAID
  • Paul Gottlieb, PhD, Dept. Microbiology and Immunology, City College of New York, NY

Contact

For more information, email schneerr@exchange.nih.gov.

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