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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 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
Joanna Kubler-Kielb, PhD, Research Fellow
Zhigang Jin, MD, Research Fellow
Zuzanna Biesova, PhD, Postdoctoral Fellow
Patrick Claude, PhD, Postdoctoral Fellow
Andres Dulcey, PhD, Postdoctoral Fellow
Paul Santacroce, PhD, Postdoctoral Fellow
C. Goran Ekborg, PhD, Oak Ridge Fellow
Jerry Keith, PhD, Oak Ridge Fellow
Ariel Ginzberg, PhD, Visiting 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, BSc Eng, 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

Photo of John B. Robbins, M.D.
Photo of Rachel Schneerson, M.D.
To achieve our overall objective of developing vaccines for infectious diseases, especially those of children, we study, uncover, and re-evaluate clinical, epidemiologic, and immunologic data. We evaluate investigational vaccines suitable for clinical study in experimental animals and then submit them to the Institutional Review Board and the FDA for evaluation of their safety and immunogenicity in adults, children, and infants, and, finally for efficacy. Surface polysaccharides of Gram-negative pathogens, capsules, or lipopolysaccharides (LPSs) are essential virulence factors and protective antigens. Immunogenicity of polysaccharides can be improved by binding to carrier proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and may serve as carrier proteins.

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

Using influenza viruses as templates for reverse transcriptase–polymerase chain reaction (RT-PCR) gene amplification or genomic sequencing data, we have produced unique recombinant hemagglutinin proteins (rHAs) from the four 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; H5N1 A/Bar-headed Goose/Qinghai/1A/2005 Clade 2, subclade 2. We designed the protein constructs to represent the mature configuration of the HA protein with the amino acid domain spanning the viral membrane deleted from the carboxyl terminal. Using Ni-ion chelating chromatography, we replaced the domain with a Gly3X-His6X tag to facilitate purification of the expressed rHA. We produced rHA from the four constructs in E. coli strains BL21 (DE3) or Rosetta 2 (DE3) and purified it from isolated inclusion bodies by solubilization of the inclusion body protein in urea and subsequent Ni-ion column chromatography. We used the construct derived from the A/Vietnam/1203/2004 H5N1 influenza virus for further studies, refolded solubilized rHA by rapid dilution into a refolding buffer and extended dialysis, and concentrated it by using a spin-filter method. By treating the purified rHA with formalin, adsorbing it onto alum or using both procedures, we prepared various vaccine candidates and injected them into mice. Sera from mice injected three times with rHA showed hemagglutinin inhibition titers of 40 and 80 or higher, suggesting that rHA can produce a protective immune response against influenza virus infection (the FDA requires a titer of 40). Our preliminary data suggest that our rHA vaccine may be produced in three to four weeks and formulated with alum; it can induce IgG anti-HA levels consistent with those recommended by the FDA for new vaccines against pandemic influenza.

Recent reports in the literature suggest that antibodies to the exposed N-terminal 23 amino acids (M2e) portion of the mature matrix 2 protein (M2) may ameliorate influenza 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 NA, 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, which explains why the protein has not been considered a vaccine target. However, recent studies in a mouse model show 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 our approach to using the M2e peptide as a vaccine, we chemically synthesized the 23–amino acid peptide and bound it to a genetically detoxified diphtheria toxin (DT-H21G) via thioether linkages; we then tested the conjugates in mice. ELISA showed high anti–M2e levels after the third injection, with or without alum. We also observed an immune response against the DT-H21G carrier. Our preliminary results suggest that this vaccine candidate may induce immunity against heterologous strains of influenza A virus.

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

H. influenzae type b (Hib) is the cause of most systemic infections by the Haemophilus influenzae pathogen. A critical level of serum IgG anti–Hib CPS (capsular polysaccharide) confers type-specific immunity by complement-mediated bacteriolysis. Herd immunity followed widespread use of this Hib conjugate vaccine, and the near-elimination of Hib led to speculation that other 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 under 4 years of age.

Hi can be divided into three virulence groups of two, related to the structures of their CPS: types a and b, the most virulent types, are composed of a neutral sugar, an alcohol (ribitol), and a phosphodiester; types c and f are composed of an N-acetylated amino-sugar, a monosaccharide, and a phosphodiester; types d and e have a repeat unit of an N-acetylglucosamine and N-acetylmannosamineuronic acid. Hib and Hia exhibit greater resistance to the bactericidal effects of complement alone than do the other four types. Challenge of infant rats showed that the 50 percent Infective Dose (ID50) for bacteremia of both Hib and Hia was several orders of magnitude lower than that of the other types. Intranasal challenge of infant rats with type b or a strains resulted in 55 to 90 percent bacteremia with type b and 35 percent with type a strains. The other types were not invasive.

Rates of Hia disease have been constant in the United States regardless of Hib vaccination. From 1998 to 2002, the Emerging Infections Program of the Centers for Disease Control conducted active laboratory- and population-based surveillance for Hi disease in a total population of approximately 35 million. 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, who have an annual incidence of Hia meningitis of 254 cases per 100,000 children. Hammitt et al. (Pediatr Infect Dis J 2005;118:e421) reported an outbreak of invasive Hia disease among Native Alaska infants. During a six-month period in two nearby villages, five cases of Hia were documented. The number of cases is too low for a randomized, double-blinded and controlled trial, although there is precedent for adding serotypes within a species to a vaccine without evidence for efficacy (viz., based on safety and immunogenicity, several pneumococcal serotypes, meningococcal groups Y and W135, and poliovirus type 2 were licensed). The increasing number of reports of Hia invasive diseases suggest that the development of an Hia conjugate is warranted.

The structural, experimental, and clinical properties of Hia CPS closely resemble those of type b. To provide a vaccine against Hi type a and several other pathogens, we investigated the feasibility of developing a single vaccine. In particular, we elucidated the structures of the six CPS types—a, b, c, d, e, and f—of Hi. Methods for conjugating type b CPS to a protein are applicable to Hia. D-1,5-ribitolphosphate is a constituent of the CPS of both 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 the antibodies induced in mice by this PS 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 two-week intervals at 2.5 g per 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. The bactericidal activity of the conjugates is under study.

Fekete A, Hoogerhout P, Zomer G, Kubler-Kielb J, Schneerson R, Robbins JB, Pozsgay V. Synthesis of octa- and dodecamers of D-ribitol-1-phosphate and their protein conjugates. Carbohydr Res 2006;341:2037-48.

Jin Z, Romero-Steiner S, Carlone GM, Robbins JB, Schneerson R. Haemophilus influenzae type a infection and its prevention. Infect Immun 2007;75:2650-4.


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. It is common among military recruits in field conditions, refugees, and patients in institutions. Shigellae are the most common cause of dysentery in children, leading to stunted growth. Vaccine development has been hindered because Shigellae are pathogens for and inhabitants of humans only. Even though the causative organism was discovered over 100 years ago, there is still no licensed vaccine against shigellosis. The World Health Organization has declared the prevention of shigellosis a top priority; it is the goal of this project.

Surface polysaccharides of pathogenic bacteria, including CPS or the O-specific polysaccharide (O-SP) of LPS, serve as both essential virulence factors and protective antigens. Covalent binding of these saccharides to medically useful proteins to form conjugates both increases their immunogenicity and endows them with T-cell dependence, making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant nontoxic P. aeruginosa exoprotein A (rEPA) exhibited an efficacy of over 70 percent in young adults exposed to attack rates of 6 to 14 percent. 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 but did not show efficacy. Antibody levels increased in a similar ratio as for adults, but the levels achieved by the conjugates were lower. Using Caco-2 and HeLa cells, we studied the effect of the immune sera and IgG isolated from the subject children on Shigella invasion into epithelial intestinal cells in vitro and found that 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). We injected mice twice, using either the same or a different carrier the second time. For S. flexneri 2a, the TT conjugate injected twice induced higher (greater than 4-fold) antibody levels. For S. sonnei, the TT conjugate followed by the rPA conjugate induced significantly higher levels than the other combinations. We are investigating the effect further.

Modification of O-SP by glycosylation or acetylation is important for O-SP’s antigenic specificity and immunogenicity. Thus, we investigated the degree of O-acetylation and the localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP. On the basis of these studies, we assigned the following structure of the repeat unit:

Modification of O-SP by glycosylation or acetylation is important for O-SP’s antigenic specificity and immunogenicity. Thus, we investigated the degree of O-acetylation and the localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP. On the basis of these studies, we assigned the following structure of the repeat unit:

Chowers Y, Kirschner J, Keller N, Barshack I, Bar-Meir S, Ashkenazi S, Schneerson R, Robbins J, Passwell JH. O-specific polysaccharide conjugate vaccine-induced IgG antibodies prevent invasion of Shigella into Caco-2 cells and may be curative. Proc Natl Acad Sci USA 2007;104:2396-401.

Kubler-Kielb J, Vinogradov E, Chu C, Schneerson R. O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a. Carbohydr Res 2007;342:643-7.

Haemophilus ducreyi and Bordetellae

We are investigating the structural characteristics of LPS and lipooligosaccharides (LOS) of human pathogens. We use the resultant information to bind the detoxified O-SP to carrier proteins and to develop experimental vaccines. We are particularly interested in both developing conjugation methods with wider applicability and formulating potential veterinary vaccines.

Bordetellae. Bordetellae are Gram-negative bacilli that cause respiratory tract infections in mammals and birds. B. pertussis, B. parapertussis, and B. bronchiseptica are clinically important. B. pertussis vaccines have been successful 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 the chemical, serological, and immunological properties of B. bronchiseptica and B. parapertussis O-SP obtained by different degradation procedures. One type of the B. parapertussis and two types of B. bronchiseptica O-SP were recognized based on their non-reducing end saccharide structure; we observed no cross-reaction between the two B. bronchiseptica types. Competitive inhibition assays showed the immunodominance of the nonreducing end of these O-SPs. We used two new methods to prepare conjugates of B. bronchiseptica and B. parapertussis O-SP with an amino-oxylated protein—one based on the Kdo residue exposed by mild acid hydrolysis of the LPS and the other on 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. Our methodologies can be applied to the preparation of LPS-based vaccines against other Gram-negative bacteria.

H. ducreyi. We studied the LOS of two strains of Haemophilus ducreyi, which is the causative agent of chancroid. The carbohydrate part of the LOS consists of either nine or six sugars terminating with partially sialylated lactosamine or lactose, respectively. Repeated subcutaneous immunization of mice with 2.5 to 5 µg of purified LOS from either strain induced IgG responses, mostly to the inner core region. We detected only low levels of antibodies to the commercially obtained neolactotetraose or sialyl-neolactotetraose (both contain the terminal lactosamine structure of the LOS). Antibodies induced by LOS injection did not bind to human glycolipids with terminal (sialyl)lactosamine structures but did bind to Lipid A or Lipid A-Kdo-Kdo saccharides, as assayed by immune thin-layer chromatography. The sera bound weakly to their homologous bacteria and were weakly bactericidal. Mice immunized with neolactotetraose or sialyl-neolactotetraose conjugated to human serum albumin produced low levels of anti–LOS antibodies. To preserve the entire saccharide part of LOS in the conjugate, we linked the 3-deoxy-D-manno-octulosonic acid (Kdo) present on the terminal reducing end of the oligosaccharide to an amino-oxy group of a bifunctional linker bound to carrier protein. The conjugates induced low levels of anti–LOS, mostly to the inner core; bound weakly to the homologous bacteria; and exhibited low bactericidal activity.

Preston A, Petersen BO, Duus JA, Kubler-Kielb J, Ben-Menachem G, Li J, Vinogradov E. Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides. J Biol Chem 2006;281:18135-44.

Vaccine development for group B Neisseria meningitidis and Escherichia coli K1

Vaccine development against group B meningococcal (GBM) meningitis is complicated by its CPS—an α-2-8-linked poly-sialic acid that is identical to the surface saccharide of the K1 CPS of E. coli as well as to many host structures. Despite effective antibiotic and supportive therapy, the mortality and morbidity of systemic infections, especially of meningitis caused by GBM and Escherichia coli K1, remain unacceptably high. GBM continue to cause epidemics and outbreaks throughout the world, and E. coli K1 is a major cause of neonatal meningitis and kidney infections. We aim to examine evidence for the association between polysaccharide (PSA) antibodies and autoimmune disease.

Meningococci are classified into serogroups according to their CPS. Of the 13 reported CPS groups, five—A, B, C, W135, and Y—cause almost all meningococcal disease. These CPSs are essential virulence factors because they inhibit the protective actions of complement. In addition, they are protective antigens in that a critical level of serum IgG CPS antibodies specifically induces both complement-mediated lysis of groups A, C, W135, and Y and opsonophagocytic killing of GBM. There are effective CPS-based vaccines for groups A, C, W135, and Y but not for GBM or E. coli K1. GBM cause a much larger number of infections in infants and young children than do those of groups A, C, W135, and Y.

Although PSA antibodies bind to many fetal and adult tissues in vitro, there is no evidence suggesting in vivo binding or associated pathology. We have therefore initiated efforts to develop vaccines that use non-capsular antigens, including outer membrane proteins, LPS, iron-binding proteins, and other antigens identified by examination of 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 would be useful for E. coli K1. Based on the performance of the Haemophilus influenzae type b, Salmonella typhi (Vi), and pneumococcal and group C meningococcal (GCM) vaccines, we developed a PSA conjugate that induced protective levels of serum IgG anti–PSA that is simple to produce, easy to standardize, and should be close to 100 percent effective at all ages. We have confirmed its performance in laboratory mice and primates.

Few studies have compared severity of infection and outcome among different meningococcal serogroups. Furthermore, published studies of sequelae in meningococcal patients make no mention of autoimmune diseases, such as Guillain Barré, multiple sclerosis, and so forth. Our review shows equal or lower rates of autoimmune disease in nearly every category of sequelae and of mortality associated with GBM meningitis versus the sequelae and mortality associated with other meningococcal serogroups. Given 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 was diagnosed in 2,984 individuals; the control population of 914 patients had group C meningococcal meningitis. Ratios of incidence rates of autoimmune disease served as measures of relative risk. Patients with GBM meningitis, in comparison to people either with group C meningococcal meningitis or no history of meningococcal meningitis, had no increased risk of autoimmune disease. A similar study in Iceland showed similar results. These findings suggest that systemic infection with GBM meningitis is not associated with autoimmune diseases in humans for up to 31 years after meningococcal disease.

Howitz M, Krause TG, Simonsen JB, Hoffmann S, Frisch M, Nielsen NM, Robbins JB, Schneerson R, Mølbak K, Miller MA. Lack of association between group B meningococcal disease and autoimmune disease. Clin Infect Dis 2007;45:1327-34.

Stein DM, Robbins J, Miller MA, Lin FY, Schneerson R. Are antibodies to the capsular polysaccharide of Neisseria meningitidis group B and Escherichia coli K1 associated with immunopathology? Vaccine 2006;24:221-8.

Peptide-protein conjugate vaccines

B. anthracis. B. anthracis 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). PA is the toxin that binds to mammalian cells. The capsule is composed of poly-d-gamma-glutamic acid (PGA) and, by itself, is non-immunogenic and has no clear protective effects. The PA-based licensed vaccine is safe and protective but has limitations that justify the development of improved vaccines. We sought to induce capsular antibodies as a potential means to expand the immunity of available anthrax vaccines.

We isolated a recombinant PA from a non-capsulated strain and observed that, in mice, several formaldehyde-treated and/or alum-adsorbed formulations were immunogenic. Our ongoing clinical evaluation of these formulations has shown that 150 18- to 45-year-old volunteers exhibited no serious adverse effects. To make a conjugate vaccine, we isolated the capsule from a non-toxic strain and bound the PGA or corresponding synthetic peptides to BSA, rEPA, rPA, or tetanus toxoid. Additional conjugating methods that used thioether, hydrazone, and oxime linkages between the PGA and the proteins, with active groups at the C or N termini, yielded immunogenic conjugates with no statistical difference. The induced antibodies were opsonophagocytic. Peptides 10- to 20-mer in length or 10 to 15 mole PGA per mole protein were the most immunogenic. The use of alum increased PA antibody levels with little effect on anti–PGA levels. With the goal of preparing humanized monoclonal antibodies to PGA, we immunized chimpanzees—one with PA-PGA and another with TT-PGA—at 10 µg PGA per animal. Both chimpanzees responded with antibodies to both vaccine components. We obtained higher anti–PGA levels in the TT-PGA–injected animal. We prepared PGA-specific IgG1 and IgG3 from these animals.

Plasmodium falciparum. Malaria ranks as one of the world’s top three deadliest diseases (approximately 300 million cases per year). P. falciparum causes the most severe form; 1 to 3 percent of the parasites are highly virulent, causing severe and cerebral malaria and the death of about 2 million people annually (90 percent of whom are young children). There is no preventive vaccine, and malarial parasites are becoming resistant to antimalarial drugs. The most studied experimental malaria vaccine candidates are the circumsporozoite protein (CSP), expressed extracellularly on the sporozoite, and various forms of the CSP’s synthesized repeat unit NANP. In setting out to develop potential vaccine candidates, we formulated vaccines that were safe and immunogenic but poorly protective, even when administered with adjuvants. Based on our studies with peptides of the B. anthracis capsule, we synthesized peptides consisting of four repeat units of NANP, bound them to carrier proteins by a scheme suitable for humans, and administered them to mice without adjuvants to study their immunogenicity. The peptides induced high levels of antibodies, with circumsporozoite-neutralizing activity roughly correlated to antibody levels measured by ELISA.

In an effort to develop a transmission-blocking vaccine, we bound Pfs25, a low–molecular weight protein, which is non-immunogenic by itself, either to itself or carrier proteins by several methods: amide, hydrazone, or thioether linkages. On evaluating the immunogenicity of the conjugates in mice, we found that all conjugates induced anti–CSP, with booster responses upon re-injection. Remarkably, at three and seven months after immunization, the serum antibody levels were higher than at one 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, and the transmission-blocking activity of immune sera correlated with antibody levels measured by ELISA.

Kubler-Kielb J, Liu TY, Mocca C, Majadly F, Robbins JB, Schneerson R. Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-d-glutamic acid-protein conjugates. Infect Immun 2006;74:4744-9.

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;104:293-8.

Protein and polysaccharide conjugate vaccines to enteric diseases

Salmonella typhi. The Vi CPS of S. typhi is a licensed typhoid vaccine. To improve its immunogenicity in young children, we conjugated the Vi to a recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A Phase 3 trial of the Vi-rEPA in 11,600 Vietnamese 2- to 5-year-olds showed an efficacy of 89 percent at 47 months. A Phase 2 trial in 301 infants started in 2006. Vi-rEPA is injected concurrently with DPT at 2, 4, 6, and 12 months. Controls receive Hib-TT and DTP or DTP alone. We will compare IgG anti-Vi levels to those elicited in the Phase 3 trial. A 10-year follow-up of adults injected once with Vi-rEPA in the Phase 1 study showed an IgG anti-Vi level 20-fold higher than the proposed protective level. We are comparing the duration of serum anti-Vi IgG in children immunized with Vi-rEPA eight years ago with serum levels measured four years ago and serum levels of unimmunized controls from another village.

Salmonella paratyphi A. Salmonella paratyphi A is the second most common cause of enteric fever in developing countries. In our Phase 1 and 2 studies, S. paratyphi A O-SP conjugated to TT proved to be safe and immunogenic in adults, teenagers, and toddlers. We constructed a mutant S. paratyphi A by replacing the LPS elongation chain length–regulator wzz gene with that of E. coli K12. We prepared a conjugate vaccine of the elongated O-SP and plan to evaluate its immunogenicity in mice.

E. coli O157. E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. Our Phase 2 trial of O-SP-rEPA conjugate in 2- to 5-year-olds (at Carolina Medical Center) showed the vaccine to be safe and immunogenic and capable of eliciting bactericidal antibodies; 98 percent of the probands demonstrated a 4-fold rise in IgG anti–LPS at 6 months. A major virulence factor of E. coli O157 is the Shiga toxin II. Alison O’Brien constructed a mutant toxoid conjugated with O-SP for enhanced protection, which is now under study.

Enterotoxigenic E. coli. Enterotoxigenic E. coli (ETEC) is the most common cause of diarrhea in developing countries. ETEC secretes two exotoxins: heat-labile toxin (LT) and heat-stable toxin (ST, a polypeptide of 19 amino acids). While LT is immunogenic in humans, its efficacy against infection has not been demonstrated. ST is small, non-immunogenic, and difficult to purify. We treated a non-toxic mutant recombinant LT, produced by John Clements, with formalin. In mice, the mutant induced high levels of IgG anti–LT and caused dose-dependent swelling at the injection site. In collaboration with Donald Robertson, we purified the ST to make it suitable for conjugation with LT or other proteins.

Vibrio cholerae O1. V. cholerae O1 remains a major health problem on the Indian subcontinent and in Africa. Field studies showed that the vibriocidal activity of antisera is directed toward the LPS. In our Phase 1 trial, the O-SP conjugates elicited high levels of IgG anti–LPS with vibriocidal activity. Chemical synthesis of O1 V. cholera O-SP is under way.

Campylobacter jejuni. C. jejuni infection is common and may cause serious complications such as Guillain-Barré syndrome, possibly as a result of the structural similarity between gangliosides and the LOS of C. jejuni. Protein conjugates of type 2 LOS or of the de-acylated LOS elicited anti–LOS IgG and bactericidal antibodies. We analyzed 28 isolates of C. jejuni from pediatric patients in Israel for serotype distribution and found the distribution to be diverse. Some isolates contain sialic acid in their LOS and bind to human ganglioside antisera.

Rotavirus. Rotavirus infection is the most common cause of infantile diarrhea worldwide. Current vaccines are reassortant live oral vaccines. One such vaccine, Rotashield, was withdrawn from the market after a suspected increase in intussusception in recipients. We designed parenteral vaccines based on the capsid proteins VP8 and VP7. Mice injected with conjugate capsid proteins exhibited neutralizing antibodies against rotavirus of the homologous (P4) and heterologous (P8) serotypes.

Ahmed A, Li J, Shiloach Y, Robbins JB, Szu SC. Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2-5-year-old children. J Infect Dis 2006;193:515-21.


Szu SCS, Konadu E, Robbins JB (2007). Vaccines against Escherichia coli, Australia Patent. Patent number 767047. Accepted.

Szu SCS, Konadu E, Robbins JB (2007). Vaccines against Escherichia coli, USA Patent. Patent number 7,247,307. Accepted.

Synthetic glycoconjugate vaccines against human pathogenic bacteria

Surface-exposed saccharides of human pathogens serve as both virulence factors and protective antigens. Such saccharides include CPS, LPS, and cell wall polysaccharides; they may vary in size and complexity. We are studying synthetic chemical approaches to bacterial surface–exposed saccharide structures for use in experimental vaccines.

Synthetic vaccine against Shigella dysenteriae Type 1. Our approach to vaccine development against Shigella is based on the hypothesis that serum IgG antibodies to the O-SP domains of the LPS of the organism confer immunity to the pathogen. We observed that, in mice, synthetic oligosaccharides corresponding to the tetrasaccharide repeating unit of the O-SP of S. dysenteriae type 1 covalently linked to human serum albumin elicited IgG anti–O-SP. The antibody levels were a function of both the saccharide’s chain length and its loading on the protein. The conjugates elicited significantly higher levels of IgG anti–O-SP than conjugates prepared with the O-SP from the bacteria. We evaluated the influence of the non-reducing terminal monosaccharide on the serum antibody response to the conjugates. We prepared synthetic oligosaccharides composed of hexa- to tridecasaccharide fragments of the native O-SP with, at their termini, one of the four monosaccharide residues that constitute the O-SP repeating unit and bound the saccharides to BSA by single-point attachments. The conjugates contained an average of 19 saccharide chains per BSA molecule. The synthetic oligosaccharides inhibited the binding of serum raised against whole-bacterial LPS but to a lesser extent than did native O-SP. Conjugates with N-acetylglucosamine (10-mer) or galactose residues (7- and 11-mers) at their non-reducing termini elicited the highest anti–LPS levels. To test the effect of different carriers, we bound hexa- to hexadecamer fragments of the O-SP to diphtheria toxoid at an average of 7 to 8 copies but found no significant differences.

As part of our synthetic vaccine project, we synthesized a tripalmitoyl cysteine derivative (Pal3Cys) that has been shown to have adjuvant properties in synthetic glycoconjugates containing covalently linked Pal3Cys. Our derivative is equipped with a linker that allows its covalent binding to proteins under mild conditions. Analyses of the derivative are under way.

Borrelia burgdorferi. Borrelia burgdorferi, the causative agent of Lyme disease, expresses on its surface a unique glycolipid that is composed of galactosyl-glycerol with palmitoly and oleoyl groups at the primary and secondary hydroxyl groups. Because of its surface location on the bacterium, this unusual glycolipid may be a candidate for vaccine development. The natural glycolipid is slightly immunogenic, and it is likely that covalent attachment to an immunogenic protein would enhance is immunogenicity. We studied chemical synthesis of the glycolipid in a form that allows its covalent attachment to proteins. Starting from commercially available R and S isopropylidene glycerols, we used a multistep synthesis to prepare an advanced derivative that features a latent aldehyde group at the end of the palmitoyl moiety. Using the reverse micelle technique, current experiments are aimed at conjugating the lipid to carrier proteins.

Synthetic organic methods development. To a large extent, the success of oligosaccharide synthesis depends on the availability of versatile O-protecting groups that are stable under a variety of conditions and that may be chemo-selectively removed by mild treatment, and thus permitting easy purification. To improve the efficiency of intermediate separation, we tested the applicability of fluorinated protecting groups. These moieties render the compounds amenable to purification with the use of adsorbents that selectively bind to fluorine-containing intermediates while not adsorbing those that contain conventional protecting groups. We are planning to use these techniques to synthesize oligosaccharides to be used as vaccine components.

A benzyl group is one of the most frequently used protecting groups in oligosaccharide synthesis. Despite use of the groups for decades, a mild method for its introduction has yet to be developed. We have prepared two potentially useful benzylating reagents, namely, benzyl N-phenyltrifluoroacetimidate and S-benzyl-mercaptobenzoxazole, and are currently evaluating their benzyl-donor ability.

Pozsgay V, Kubler-Kielb J. Synthesis of carbohydrate antigens related to Shigella dysenteriae type 1 and their protein conjugates. In: Demchenko AV, ed. Frontiers in Modern Carbohydrate Chemistry. ACS Symposium Series 2007;960:238-52.

Pozsgay V, Kubler-Kielb J. Synthesis of an experimental glycolipoprotein vaccine against Lyme disease. Carbohydr Res 2007;342:621-6.

Pozsgay V, Kubler-Kielb J, Schneerson R, Robbins JB. Effect of the nonreducing end of Shigella dysenteriae type 1 O-specific oligosaccharides on their immunogenicity as conjugates in mice. Proc Natl Acad Sci USA 2007;104:14478-82.

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

Amino sugars are common components of bacterial polysaccharides that are either N-acetylated forms (for example, Neisseria meningitidis group A and Shigella dysenteriae type 1) or sugars with a free amino group (Shigella sonnei). As part of a program of vaccine development based on these polysaccharides, we are interested in extending the use of nitrogen nuclear magnetic resonance (NMR) parameters for the structural, stereochemical, and conformational analysis of these materials. In our previous work, we used the inverse-detected, heteronuclear single quantum, multiple-bond correlation (HSQMBC) technique to measure the 15N–1H coupling constants of a suite of organic-soluble amino sugar derivatives. We selected the derivatives for the initial study because (1) their expected long relaxation times appeared to offer the greatest chance of success for application of the multipulse HSQMBC technique and (2) the available derivatives contained 15N and 1H nuclei in several different stereochemical orientations. We correlated the magnitudes of the vicinal 1H–15N coupling constants with the geometry of the coupled nuclei in the conformations of the amino sugar models, as determined by molecular dynamics/mechanics computations. Non-linear regression of the vicinal 1H–15N coupling constants to the HCCN dihedral angles determined by molecular dynamics led to the definition of a new Karplus equation: 3JHCCN = 3.1 cos2 phi = 0.6 cos phi + 0.4, which describes the dependence of the vicinal, 3JHCCN coupling constant on the HCCN dihedral angle phi in amino sugars derivatives.

During the past year, we extended the above measurements to the common amino sugars in aqueous solution—conditions that correspond more closely to physiological conditions than does solution in the previously studied organic solvents. The common amino sugars occur frequently in bacterial polysaccharides, including N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and 3-acetamido-3-deoxy-d-glucose, and in the case of the free amino sugars glucosamine, mannosamine, and galactosamine, which we studied as their stable hydrochloride salts. We adopted the recently published CPMG-HSQMBC NMR technique (Kövér et al., J Magn Reson 2006;181:89) for these measurements. The technique involves repetitive refocusing of nuclear magnetization by the Carr-Purcell-Meiboom-Gill procedure to suppress evolution of proton magnetization, thus affording antiphase HSQMBC NMR spectra of higher quality than the older method.

We tested several polar and semipolar solvent systems, including deuterium oxide, 10 percent deuterium oxide:90 percent water, methylsulfoxide-d6, and 107:1 w/v methylsulfoxide-d6:trifluoroacetic acid. When we began the study, it was not known whether dipolar relaxation of the 15N nucleus by an attached proton would be required for application of the HSQMBC method. It was therefore unclear whether deuterium oxide could be used as a solvent. In deuterium oxide, all OH and NH protons are expected to undergo deuterium exchange, thus removing the strong dipolar relaxation mechanism provided by the protons. However, the results of the study revealed that dipolar relaxation by 1H directly bonded to 15N is not required for success of the experiment; therefore, use of deuterium oxide is entirely feasible.

In our previous work, we found that determination of the vicinal 1H–15N coupling constants from the HSQMBC NMR spectra is easiest if the inverse-detected 1H multiplets have minimum multiplicity, a situation that obtains when the sugar hydroxyl protons are in rapid chemical exchange or have been exchanged with deuterium. In the present study, we observed that use of deuterium oxide as a solvent fulfills both conditions. We found that a solvent containing 1 part trifluoroacetic acid in 107 parts of methylsulfoxide-d6 catalyzed rapid hydroxyl proton exchange, thus removing the hydroxyl proton coupling constants from the multiplets and facilitating interpretation of the spectra.

Interpretation of the HSQMBC NMR spectra requires complete 1H and 13C assignments for the amino sugars. Therefore, part of our project calls for 2D COSY (two-dimensional COrrelation SpectroscopY) and 2D HSQC (Heteronuclear Single-Quantum Correlation) spectra to obtain definitive 1H and 13C assignments in order to analyze, respectively, the 1D 1H and 13C NMR spectra of mixtures of the pyranose anomers of the amino sugars. Analysis of the NMR data is under way, and we expect that the results will provide a clearer understanding of the effects of the orientation of electronegative substituents on the 1H–15N coupling constants, along with additional information on their dihedral angle dependence.

Coxon B. A Karplus equation for 3JHCCN in amino sugar derivatives. Carbohydr Res 2007;342:1044-54.

NMR verification of structures of bacterial saccharide precursors for vaccines

High-resolution nuclear magnetic resonance spectroscopy is a powerful method for analyzing the conformations, molecular structures, purity, and stereochemistry of carbohydrates, including the monosaccharide, oligosaccharide, and polysaccharide types that are of prime interest in bacterial vaccine development. We provide analytic NMR support as needed for current vaccine projects involving the characterization of saccharide structure and purity. Our general goals are to determine, verify, or characterize saccharide structures and purity by high-resolution NMR spectroscopy. We investigate new vaccine-related substrates as they are developed.

Using NMR at 500 MHz, we studied three fractions of O-SP isolated from Shigella sonnei and verified the structure of the polysaccharide with 2D COSY, HSQC, and TOCSY (Total Correlation Spectroscopy) methods, in conjunction with 1D 1H and 13C NMR and 1H-coupled 13C NMR, for confirmation of the anomeric configuration of the sugar residues. We also inspected for correct interpretation and authenticity the 300 MHz 1H NMR spectra of about 40 batches of monosaccharide intermediates and linkers directed to the preparation of a conjugate vaccine for Vibrio cholera. With the degree of spectral dispersion (spreading of the NMR spectrum) and spectral resolution (narrowness of the resonances) limited at 300 MHz, we supplemented the NMR data for the Vibrio cholera intermediates with 10 sets of 1H and/or 13C NMR data measured at 500 MHz. These spectra provide more detailed structural information and spectral assignments than are available from 300 MHz NMR spectra; thus, we were able to detect, for example, the presence of any small, long-range 1H1H coupling constants. Using high-resolution NMR spectroscopy at 500 MHz as well as 1H NMR, 13C NMR, and 2D HSQC NMR to correlate and assign the 1H and 13C chemical shifts, we were able to characterize the structures of five chromatographic fractions of Klebsiella pneumoniae type 2 CPS. The NMR spectra provided information on the general types of structures of the fractions’ components as well as their purity while permitting comparisons with the literature’s data on related materials.

Isolation and NMR elucidation of three saponins from Blighia sapida

Saponins are steroid glycosides, steroid alkaloids (steroids with a nitrogen function), or triterpenes; they 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 can cause urticaria. We set out to isolate three saponins from Blighia sapida and to determine their structures by using high-resolution NMR spectroscopy and mass spectrometry.

QS-21 is a complex saponin that has been used as a vaccine adjuvant and thus has stimulated interest in saponins as a general class of compounds. 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 now 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 comprising 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 the determination of structure included 1D slices of 2D TOCSY data sets to generate separate sub-spectra for the individual monosaccharide residues, thereby allowing identification of sugar type by measurement of vicinal 1H1H coupling constants. We also used 2D HMBC experiments that yielded 1H/13C through-bond connectivities that defined the nuclear neighbors in the structures as well as the sugar linkages and positions of acetyl substitution. 2D ROESY 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 by 2D 1H-coupled HSQC spectra, which also allowed identification of equatorial or axial protons in the sugar as well as determination of the anomeric configuration of each sugar by measurement of 1JC-1,H-1 coupling constants.

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

Heparin and heparan sulfates (H/HS) constitute a class of highly sulfated glycoamino-polysaccharides found in cell membranes. Their sugar modifications and sulfate density are structurally diverse, providing H/HS with a large number of unique sequences and accounting for their ability to modulate the functions of diverse proteins and systems in normal and disease processes (cell growth, secretion, development, blood coagulation, and infections by viruses and other pathogens). Owing to the diversity of H/HS sequences, libraries of unique H/HS sulfated oligosaccharides (oligoS) are not readily obtainable. To obtain a macrocombinatorial heparin-mimetic family (library) of sulfated oligosdaccharides based on the structure-function model of anticoagulant heparin (Rosenberg RD et al.), we selected as an antithrombotic the German pharmaceutical SP54, a chemically sulfated natural xylan developed in the 1960s, that consists of sulfated glucuronyl-oligoxylans (S-oligoS) that mimic heparin’s multiple biological actions.

SOLIS, potential HIV-1 viral entry inhibitor. The successful long-term control of HIV viral load requires inhibitors other than those that function as protease or reverse transcriptase inhibitors against viral components. We showed that the in vitro inhibition of HIV-1 cytotoxicity and syncytium-formation were each governed by structural specificity as well as by separability from the anticoagulant S-oligoS of the pharmaceutical—essential indicators of the usefulness of S-oligoS for drug development. We devised clinically acceptable procedures for the preparation of a highly active HIV-1 virus fusion inhibitor free of antithrombin toxicity (PK II, SOLIS) and are completing a clinical preparation of SOLIS for a Phase I i.v. administration trial. SOLIS inhibits binding to and entry of virus into target cells (e.g., by inhibiting CD4 cell adherence to gp120-coated tissue culture plates [binding assay] and by cell-killing [cytopathology assay]; it is highly active against syncytium formation of HIV-1 [gp41] with CD4 cell membrane [fusion assay]). Given that heparin affects conformation of proteins to modulate their biological function, a possible mechanism of SOLIS’s action is inhibition of the conformational changes of gp41 associated with fusion progression.

It has long been known that S-oligoS is poorly absorbed by oral administration. Our strategy would use i.v. administration to achieve the required blood level as assessed by IC 50s found in bioassays in vitro. A relatively large amount of starting source would be required. We are investigating an FDA-licensed heparin-mimetic, similar to the European-made SP54, which might serve as a more readily obtainable source of our heparin-mimetic components. We have made two small preparations of components analogous to SOLIS (one exhibits inhibitory capacity [formazan cytopathology assay] similar to that of PK II). Viral proteins that enable HIV to attack, survive, and replicate in cells are available for in vitro study. To identify and isolate putative H/HS ligand(s), we would bind a bifunctional SOLIS probe photochemically to viral/cell proteins during virus attack under conditions of HIV-1 tissue-culture infection and would analyze isolated membrane proteins for the fluorescent probe. Such ligand(s) might be protective antigens and/or a means of identifying endogenous H/HS receptors.

Studies on combination therapy. We are preparing the antimalaria S-oligoS Cp11 to elucidate parameters for co-administration with SOLIS. We are working to determine whether the addition of Cp11 (relatively inactive against HIV-1) to PK II doses in the formazan cytotoxicity protection assay will affect the capacity of PK II to protect cells.

Heparin-mimetic sulfated oligoxylan inhibitors of malaria parasites

Our goal is to apply and expand our macromolecular family of S-oligoS to study components for their antimalarial potential. The first objective is to elucidate further a novel antimalaria drug that is a potent in vitro inhibitor of primary invasion of hepatocytes. The second objective is to test our H/HS family in a rosetting assay to identify potential S-oligoS component(s) for drug development against cerebral malaria in young children. The third objective is to elucidate potential combined therapy for combating the increased incidence and spread of HIV infection.

Heparin is known to inhibit the initial invasion of hepatocytes (and possibly red blood cells [RBCs]) by Plasmodium falciparum and the rosetting of parasitized erythrocytes (PfRBCs) and their cytoadhesion to normal RBC and the endothelium; it clears blockage of the microcirculation and ameliorates life-threatening symptoms of cerebral malaria when administered to children. We previously applied a macrocombinatorial strategy to study heparin inhibition in vitro by using our library of sulfated oligoxylans, which we prepared from an H/HS-mimetic pharmaceutical with numerous biological actions of the heparin family. We measured the capacity of P. yoelii sporozoites to invade hepatocytes and demonstrated differential inhibition of parasite invasion by S-oligoS, with the highest potency and concentration dependence residing in two S-oligoS of about 7,200 and 3,700 D with 43 and 56 percent inhibition at 3.5 and 5 micromolar doses, respectively.

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 90 percent sulfated and highly negatively charged. The motif could accommodate the subtle variations in geometry that would provide for multifunctional mimicry of the heparins. The mass of Cp 6 and 3 would accommodate the presence of 6 and 2 such motifs, respectively. Studies on inhibition of the RBC invasion stage of malarial parasites revealed that S-oligoS of a mass class less than 4,500 exhibited markedly low inhibitory capacity, whereas high potency was associated with S-oligoS with a mass greater than 10,000. The results suggest a difference in the molecular reactions underlying inhibition of the two parasite stages. We continue to enlarge our H/HS-mimetic S-oligoS library to generate specific antimalaria components of mass class Cp 4 and 5 (8,000 to 9,000). Recent advances in malaria research include John Sacci’s development of the first method for generating sporozoites (and other stages) in cell-free culture, which will provide enough sporozoites to complete full dose-response measurements of groups of S-oligoS in bioassays. We will resume our studies on the inhibition of sporozoites, parasitized RBC, and rosetting as a path toward stable, inexpensive heparin-mimetic antimalarials against initial infection, pathologies, and/or acute cerebral malaria. We plan to elucidate putative protein ligands by using a modified gel-shift analysis and/or to identify protein ligands by using fluorescent receptors.

Neonatal respiratory distress related to colonization with group B streptococci

Respiratory distress is a prominent clinical feature in newborns with early-onset group B streptococcal (GBS) disease. A previous study suggested that respiratory distress might be caused by pulmonary hypertension induced by GBS infection. Pulmonary hypertension reflects an increase in pulmonary vascular resistance, which impairs exchange of oxygen and carbon dioxide. It is known that infusion of live or heat-killed GBS into sheep promptly induces pulmonary hypertension, with little or no effect on systemic pressure. An increase in thromboxane A2 mediated the hypertension. Jerri Curtis and colleagues recently purified and identified phospholipids (cardiolipin and phosphatidylglycerol) from the GBS cell wall as pulmonary hypertensive compounds (Curtis et al., Proc Natl Acad Sci USA 2003;100:5087). Infusion of GBS phospholipids into baby lambs caused pulmonary hypertension. Curtis and colleagues hypothesized that patients infected with GBS may acquire a dose of bacterial phospholipids capable of causing pulmonary hypertension and respiratory distress. Other scientists demonstrated that exposure of Streptococcus mutans to penicillin induces an immediate release of phospholipids from bacteria; cardiolipin and phosphatidylglycerol constitute more than one-half of S. mutans phospholipids. Newborns who are infected with GBS or whose mothers carry GBS are usually treated with penicillin or its derivatives. Thus, antibiotic treatment may cause an increase in production and excretion of phospholipids.

We used clinical and epidemiologic data collected in the NICHD multicenter GBS study to examine a possible association among GBS colonization, penicillin treatment, and pulmonary hypertension in neonates. The prospective study, conducted from 1995 to 1999, involved 1,674 of 17,690 newborns cultured at four sites and found to be colonized with GBS. We analyzed 1,610 colonized newborns of 32 or more weeks’ gestation without early-onset disease. We compared clinical features between 1,003 lightly colonized and 607 heavily colonized newborns, focusing on rates of respiratory distress in the colonized newborns of penicillin-treated mothers versus rates of respiratory distress in the newborns of untreated mothers. Of the 1,610 colonized newborns, 8.8 percent had signs of respiratory distress within 48 hours of birth (cases) compared with 2 to 3 percent in the general newborn population. Oxygen supplementation was used in 60 percent of the cases, mechanical ventilation was required in 5 percent, and persistent pulmonary hypertension was diagnosed in 2 percent. Compared with light colonization, heavy colonization increased the rate of respiratory distress 1.73-fold and a discharge diagnosis of respiratory disorder 2.02-fold. Penicillin use during labor was associated with a 2.62-fold increase in respiratory distress in the colonized newborns.

Although experimental data have shown the effect of penicillin on the release of phospholipids from S. mutans, extrapolation of these data to clinical observation in human newborns would require an assay that measures bacterial phospholipids in the biologic specimens of GBS-colonized newborns. In collaboration with Alfred Yergey, we are developing such an assay to measure serum bacterial phospholipids. This year, we expect to begin a prospective study that relates serum bacterial phospholipid levels to respiratory distress in newborns of GBS-colonized mothers .

We undertook studies to determine the prevalence of GBS types in U.S. neonates. Our principal aim is to determine whether phylogenetic lineages of GBS vary in their ability to infect and colonize human neonates. To this end, in a prospective multicenter study, we characterized phylogenetic lineages of isolates collected from infected and asymptomatically colonized infants. We identified phylogenetic lineages by using molecular markers already developed in John Bohnsack’s laboratory and by undertaking multilocus sequence typing (MLST) that characterized lineages not identified by current molecular markers. MLST reproducibility allows the data generated in our studies to be compared with epidemiologic investigations carried out anywhere.

We studied the phylogenetic lineages of GBS isolated from infected and colonized neonates collected from the NICHD multicenter GBS study described above. We determined the phylogenetic lineage of each GBS isolate by MLST and then assigned a sequence type (ST) to each isolate. Isolates clustered into clonal complexes (CCs). We studied 899 neonatal GBS isolates, of which 129 were associated with invasive disease. Almost all invasive and colonizing serotypes Ia, Ib, and V isolates were from single clonal complexes: 353 of 383 (92 percent) type I were CC 23; 78 of 81 (96 percent) type Ib were CC 12; and 134 of 153 (88 percent) type V were CC 1. In contrast, serotype II and III strains were mostly from two clonal complexes—CC 17 and CC 19. Similar to a result previously described for CC 17 serotype III GBS, CC 17 serotype II GBS was isolated from a higher percentage of infected infants than from colonized infants. While the vast majority of invasive GBS was confined to the same lineages that colonized neonates, we found 9 invasive GBS isolates that were from lineages uncommon among colonizing isolates.

Lin FY, Troendle JF. Hypothesis: neonatal respiratory distress may be related to asymptomatic colonization with group B Streptococci. Pediatr Infect Dis J 2006;25:884-8.

Lin FY, Whiting AA, Adderson EE, Takahasi SS, Dunn DD, Azimi PH, Philips J, Weisman LL, Regan JJ, Clark PP, Rhoads GG, Frasch C, Troendle JF, Moyer PP, Bohnsack JJ. Phylogenetic lineages of invasive and colonizing strains of serotype III group B Streptococci from neonates: a multicenter prospective study. J Clin Microbiol 2006;44:1257-61.


Amina Ahmed, MD, Carolina Medical Center, Charlotte, NC
Shai Ashkenazi, MD, Schneider Children’s Hospital, Petah Tikva, Israel
Joseph A. Bellanti, MD, Georgetown University Hospital, Washington, DC
John F. Bohnsack, MD, University of Utah Health Sciences Center, Salt Lake City, UT
Dianjun Cao, PhD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
George Carlone, PhD, Centers for Disease Control and Prevention, Atlanta, GA
Yehuda Chowers, MD, Chaim Sheba Medical Center, Tel Hashomer, Israel
John Clements, PhD, Tulane University Health Sciences Center, 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, Lanspítali University Hospital, Reykjavik, Iceland
McDonald K. Horne, MD, Hematology Service, NIH Clinical Center, 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 and The Graduate School and University Center, City University of New York, Bronx, NY
Dennis J. Kopecko, PhD, Laboratory of Enteric and Sexually Transmitted Diseases, CBER, FDA, Bethesda, MD
Tyra Grove Krause, MD, PhD, Statens Serum Institut, Copenhagen, Denmark
Teresa Lagergard, PhD, Sahlgrenska Academy, Göteborgs Universitet, 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, Molecular Interactions Resource, NIBIB, Bethesda, MD
Eugene P. Mazzola, PhD, Joint Institute for Food Safety and Applied Nutrition, University of Maryland, College Park, MD
James McMahon, PhD, Molecular Targets Development Program, NCI, Bethesda, MD
Wilbur 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
Kare Mølbak, MD, Statens Serum Institut, Copenhagen, Denmark
Sophie E. Moore, PhD, London School of Hygiene and Tropical Medicine, London, UK
Alison O’Brien, PhD, Uniformed Services University of the Health Sciences, Bethesda, MD
Ainsley Parkinson, BS, Lehman College and The Graduate School and University Center, City University of New York, Bronx, NY
Andrew M. Prentice, MD, London School of Hygiene and Tropical Medicine, London, UK
Donald C. Robertson, PhD, College of Veterinary Medicine, Manhattan, KS
Sandra Romero-Steiner, PhD, Centers for Disease Control and Prevention, Atlanta, GA
John Sacci, PhD, University of Maryland, Baltimore, MD, and Walter Reed Army Institute of Research, Washington, DC
Joseph Shiloach, PhD, Laboratory of Cellular Developmental Biology, NIDDK, Bethesda, MD
Darrell Singer, MD, 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
Yimin Wu, PhD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
Alfred L. Yergey,PhD, Program in Physical Biology, NICHD, Bethesda, MD

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