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Development of Vaccines to Bacterial Diseases found Especially in Children

John B. Robbins, MD, Co-Director, Program in Developmental and Molecular Immunity
Rachel Schneerson, MD, Co-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
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
Chunyan Guo, BSc, Charles River Research Assistant
Steven W. Hunt, BSc, Research Assistant
Arthur B. Karpas, PhD, Charles River Adjunct Investigator
Jerry Keith, PhD, Oak Ridge Fellow
Joanna Kubler-Kielb, PhD, Research Fellow
Wen-Tzu Lai, PhD, Research Fellow
Jianping Li, MD, Guest Researcher
Feng-Ying (Kimi) Lin, MD, MPH, Medical Officer
Fathy D. Majadly, BSc, Senior Research Assistant
Victor C. Nelson, PhD, Senior Research Fellow
Cynthia Ng, MS, Kelly Services Research Assistant
Elizabeth Ogbonna, BSc, Charles River Research Assistant
Je-Nie Phue, PhD, Charles River Research Adjunct Investigator
Vince Pozsgay, PhD, Staff Scientist
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)

Herd immunity followed widespread use of the Haemophilus influenzae type b (Hib) conjugate vaccine, and the near-elimination of Hib led to speculation that other Haemophilus influenzae (Hi) types may emerge as causes of meningitis. In the US rates of Hia disease have remained constant despite Hib vaccination and are high especially among Native Americans. The structural, experimental, and clinical properties of Hia capsular polysaccharide (CP) closely resemble those of type b, and the increasing number of reports of Hia-invasive disease suggests that development of an Hia conjugate is warranted. Methods for conjugating type b CP to a protein are applicable to Hia. Other constructs and formulations are being studied: D-ribitol-1-phosphate is a constituent of the CPs of 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. Injected into mice the conjugates demonstrated antibodies to both Hia and Hib, with the octamer conjugate a better immunogen than the dodecamer. Some sera showed bactericidal activity against both type a and type b, correlated roughly with their ELISA values. Other constructs and formulations are being studied. A dimer of Hia CP has been prepared.

Bordetellae and Haemophilus ducreyi and Brucellae

The licensed pertussis vaccines confer incomplete efficacy on an individual basis. Induction of bactericidal antibodies would increase vaccine efficacy. Based on the concept that IgG anti-LPS provides immunity to non-capsulated Gram-negative bacteria, we studied chemical, serological and immunological properties of LPS-derived saccharides of B. pertussis and B. bronchiseptica, -reported to share the same LPS core-, obtained by different degradation procedures, and their protein conjugates.

B. pertussis and B. bronchiseptica cores were conjugated to aminooxylated BSA via their terminal Kdo. Injected into mice, both conjugates induced similar IgG anti B. pertussis LPS levels. Mutants deficient in O-SP used: 1. RB50 delta lacks the O-SP but its core structure is identical to that of the parent strain, 2. RBA2b produces LPS with no O-SP, but with repeats of the three non-reducing end core saccharides. Fractions of the B. bronchiseptica core with 1 to 4 repeats of this terminal trisaccharide bound to BSA at different densities were immunogenic in mice, the highest antibody levels were obtained by conjugates containing 10-15 saccharide chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced sera were bactericidal against B. pertussis; their titers correlated roughly with IgG anti LPS levels measured by ELISA.

Brucellosis, a zoonosis caused by various Brucella species, affects sheep, goats, deer, and more. Humans become infected by contact with animals or animal products contaminated with the organism. Symptoms in humans include fever, sweats, headaches, back pain, and weakness. Severe infections of the CNS, heart, GI tract may occur and chronic symptoms include recurrent fever, joint pain, and fatigue. The main species pathogenic for humans are B. abortus, B. melitensis and B. suis. The lipopolysaccharide (LPS) of Brucella is a major virulence factor and a potential protective antigen. The 3 Brucella species share an O-SP; a non-branched homopolymer of 4,6-dideoxy-4-formamido-a-D-mannopyranose. Conjugates of O-SPs of the 3 species and aminooxylated BSA were prepared and the B. abortus O-SP conjugate injected into mice induced IgG anti O-SP with booster responses and reacted with both B. melitensis and B. suis by ELISA.

Chancroid, a sexually transmitted genital ulcer disease is caused by Haemophilus ducreyi, producing a tri component cytolethal toxin; cdtA, cdtB, and cdtC. CdtA and C comprise the toxin component binding to host cell receptors and translocate cdtB, the catalytic toxin component, into host cell nuclei. Three obtained plasmids carrying modified forms of the cdt genes were sequenced, characterized and PCR primers amplifying the mature cdtA and C prepared and used to clone the modified genes into a protein expression plasmid. The plasmid was transformed into E. coli DH5-alpha for seed stocks and E. coli BL21(DE3) for expression. The recombinant cdtA and C proteins were purified from inclusion bodies using 6 M urea and Ni-ion affinity chromatography and characterized by re-sequence analysis, polyacrylamide gel electrophoresis and western blots.

Shigellae

Shigella sonnei and S. flexneri 2a O-SPs bound to recombinant P. aeruginosa exoprotein A were evaluated in a Phase 3 study in 1- to 4-year-olds, with each conjugate serving as a control for the other. The vaccines were safe. Immunogenicity was age-related. The S. sonnei conjugate had about 70 percent efficacy in 3- to 4-year-olds but no efficacy in 1- to 2-year-olds. There were too few cases of S. flexneri 2a infection for statistical analysis. Protection from non-vaccine types of S. flexneri, in S. flexneri 2a conjugate recipients, was noticed especially for type 6, the most common S. flexneri isolate during the study. More immunogenic vaccine candidates were prepared. Low molecular mass O-SP-core (O-SPC) fragments containing an average of 3-4 repeat units of S. sonnei O-SP were isolated and bound to carrier proteins. 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. A clinical lot of this vaccine candidate was prepared.

Peptide-protein conjugate vaccines

Bacillus anthracis. Formaldehyde-treated and/or alum-adsorbed formulations of a recombinant PA were injected 3 times, 2 months apart, followed by another injection 1 year later into adult volunteers. All formulations were safe and immunogenic. Antibody assays compared favorably with those of the licensed vaccine.

Peptides of D-gamma-glutamic acid (D-G-PGA) of B. anthracis capsule of various lengths and densities per carrier were bound to BSA, rEPA, rPA or TT. Peptides of 10 to 20-mer long and 10 to 15 mole PGA per mole protein were the most immunogenic. Chimpanzees were immunized sc with rPA-PGA or TT-PGA (10 mcg D-G-PGA/animal). Both chimps responded with antibodies to both vaccine components. Higher anti-PGA levels were obtained with the TT conjugate. Five D-G-PGA-specific Fabs were generated. Two were converted into full-length human constant region IgG1 and IgG3 monoclonal antibodies (mAbs). A single 30 mcg dose of either mAb, given to BALB/c mice 18 h before intratracheal spore challenge with the virulent B. anthracis Ames strain, conferred protection from about 40 LD-50. Also, both mAb given 8 h or 20 h after challenge provided significant protection. Thus, these anti-D-G-PGA mAbs would be useful, alone or in combination with anti-toxin mAbs, for a safe and efficacious post-exposure therapy for anthrax.

Plasmodium falciparum. The circumsporozoite protein (CSP), and various forms of its repeat unit, NANP, were the most studied experimental malaria vaccines. These vaccines were safe, but their protection was poor and of limited duration. We used two approaches to provide experimental malaria vaccines:

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. Long term studies revealed a unique property of Pfs25 bound onto itself; IgG antibody levels increased with time, peaking at around 7 months and starting to decline at 9. Antibody levels of Pfs25 conjugated to other carriers started to decline after 3 months. Adsorption of the conjugates onto alum increased further the antibody levels. Similar results were obtained with Pvs25-Pvs25 conjugates. Transmission blocking activity of Pfs25- Pfs25 immune sera correlated with antibody levels measured by ELISA.
Following earlier studies using NANP (Asn-Ala-Asn-Pro), the repeat fragment of the pre-erythrocytic parasite stage as a vaccine, we prepared 4 and 5 NANP repeats conjugated to BSA. These conjugates were immunogenic in mice, induced booster responses with corresponding high titers in IFA. The addition of a CSP T-cell epitope to the NANP repeats did not enhance anti-CSP levels or persistence. The identity of the terminal amino acid of NANP was critical; terminal Asn as NANP or NPNA, were the best immunogens. The optimal density of the peptide per carrier was around 10 with no difference between 4 and 5 repeats. Alum adsorption enhanced antibody production to both vaccine components.

The immunogenicity of an additional CSP-derived tetrapeptide, NVDP, was studied. NANPNVDP₂C, NVDPNANP₂C and NVDP₄C were synthesized, bound to BSA through thioether linkages, at 4 to 20 chains per protein and their immunogenicity evaluated in young GP mice. For all peptides, 5-9 chains per protein were optimal. The highest antibody levels were obtained using NANPNVDP but these levels did not exceed those induced by NANP only conjugates. However, combining a NANP conjugate with NVDP or NANPNVDP conjugate increased significantly anti-CSP levels with corresponding high IFA titers.

An obtained plasmid carrying a modified P vivax csp gene was sequenced. The reading frame related to the csp gene of the sequenced plasmid was characterized, and PCR primers produced to amplify the gene encoding the mature CSP, minus the signal and the carboxy-terminal GPI-anchor sequences. With these primers, the gene was amplified and cloned into a protein expression plasmid that was then transformed into host E. coli DH5-alpha and E. coli BL21(DE3). The r-CSP was purified from inclusion bodies using 6 M urea and Ni affinity chromatography. The amino acid sequence of the recombinant protein was verified by DNA sequence analysis of the new plasmid construct, and the protein characterized by SDS-PAGE and Western blot.

Protein and polysaccharide conjugate vaccines to enteric diseases

Salmonella typhi. The capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine but with limited efficacy in children less than 5 years old. To provide a vaccine for younger children, Vi was conjugated to a recombinant Pseudomonas aeruginosa exotoxin A (rEPA). Vi-rEPA had an efficacy of >90% in 2- to-5-year olds. Safety and immunogenicity of Vi-rEPA administered concurrently with the vaccines of the Expanded Program of Immunization (EPI) at 2, 4, 6 and 12 months were studied in 301 Vietnamese infants. Controls received Hib-TT +EPI or EPI only. No serious adverse events occurred in any groups. The GM IgG anti-Vi level in the Vi-rEPA group was significantly higher than in the control groups with no difference in the level of IgG antibodies to the toxoids of diphtheria, tetanus and to pertussis toxin among all groups; Vi-rEPA is suitable for routine immunization in infants. Maternal antibodies impeded infants' responses. Vi conjugated to DT had similar properties to those of Vi-rEPA.

Vibrio cholerae O1 remains a major health problem in developing countries; especially in the Indian subcontinent and in Africa. A cholera outbreak of 420,000 cases and 6,000 deaths occurred in Haiti in 2010-11, No vaccine is available to control the spread. Vibriocidal activity is directed toward V. cholerae LPS. In a phase 1 trial, V. cholerae O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. A hexamer and an octamer corresponding to the O-SP were chemically synthesized and conjugated to TT. These conjugates elicited in mice higher vibriocidal activity than the native O-SP conjugate used in the Phase I study, with the octamer- eliciting higher levels than the hexamer- conjugate. A conjugate synthesized with a heptadecamer linker was more immunogenic than the one with a nonamer linker. Linkers with various hydrocarbon or hydroxyl chains were compared for efficiency, stability, and solubility.

Rotavirus is the most common cause of infantile diarrhea worldwide. A parenteral vaccine based on capsid proteins is being designed. Recombinant capsid proteins with truncated C or N termini were expressed in E. coli and elicited neutralizing antibodies in mice and guinea pigs. Conjugation of the recombinant proteins to a polysaccharide vaccine improved protein solubility. The core region of the capsid protein is being investigated.

Enterohemorrahagic E. coli (EHEC) infections are the leading cause of E. coli deaths in developed countries. In the US, the prevalent serotype is O157H:7. EHEC strains contain the Shiga toxin (Stx) gene and may cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS). HUS is a major cause of acute and chronic kidney damage in young children and can lead to death. In a phase II study in 2-5 years old children in the US, an LPS based conjugate vaccine elicited a >10 fold rise of antibodies with bactericidal activity in 98% the children. Most EHEC infections are caused by strains secreting Stx2. The non-toxic B-subunit of Stx2 was purified in high yield by an improved recombinant technique.

Synthetic vaccines against human pathogenic bacteria

E. coli O148 is an occasional cause of dysentery, similar to the one caused by S. dysenteriae type 1. The O-specific polysaccharides of these bacteria are similar. Both are composed of tetrasaccharide repeat units, the only difference between them is the replacement of a glucose residue in the former by a galactose residue in the latter. To study cross-reactivity between these organisms, we have synthesized chemically saccharide fragments from mono- to dodeca-saccharides, equipped with a linker enabling their covalent attachment to proteins, using our oxime conjugation protocol. Conjugate-induced sera in mice reacted with the homologous and heterologous LPSs. This finding establishes cross-reactivity in both directions and supports the view that an O-specific oligosaccharide-based vaccine against one of the organisms will offer protection against both bacteria.

Borrelia burgdorferi causes Lyme disease that afflicts an increasing number of people of all ages, including children, in large areas of the US. There is no vaccine against this organism. B. burgdorferi expresses two groups of glycolipids on its surface termed BBGL-1 and BBGL-2. The BBGL-1 structure was previously defined. BBGL-2 is composed of alpha-galactosyl-glycerol bearing two fatty acid moieties on the glycerol part. To map the importance of the various structural moieties for antibody induction, we have synthesized chemically a panel of BBGL-2 analogs that contain the galactosyl moiety in alpha or beta anomeric configuration, and have the fatty acid components in alternate positions. Our findings: the anomeric configuration of the galactose moiety is not important in antibody recognition, the glycerol moiety has to bear two fatty acid moieties of which one must be an oleoyl, at either sn-position 1 or 2. Based on these findings we synthesized conjugatable derivatives of the native BBGL-2 glycolipid and attached them to BSA. The synthetic glycolipoprotein induced specific antibodies in mice. This observation forms the basis for the synthesis of our next generation BBGL-2 conjugates.

Similarly to Haemophilus influenzae type b, Haemophilus influenzae type a (Hia) causes meningitis especially in children. The capsule of Hia is composed of repeat units of glucosyl-ribitol-phosphate. We designed a chemical synthetic approach to provide an Hia vaccine. Our approach employs synthesis of a complete repeat unit followed by its polymerization: We assembled a repeat unit linked to a spacer for eventual coupling to a protein. Next, we extended the polymer chain using another repeat unit. Iterative chain extension led to a trimer of the glucosyl-ribitol-phosphate repeating unit. We are optimizing the synthetic steps to allow the preparation of higher-numbered oligomers.

Neonatal respiratory distress related to colonization with group B streptococci

Despite the decrease in the incidence of early-onset group B streptococcal (GBS) disease due to widespread use of antibiotic prophylaxis during labor, GBS remains a leading cause of neonatal sepsis. 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. Studies on the effect of GBS on pulmonary hemodynamics in animal models showed that infusion of live or heat-killed GBS into sheep promptly induced pulmonary hypertension. The identification of GBS cell wall cardiolipin and phosphatidylglycerol as pulmonary hypertensive compounds, and that exposure of Streptococcus mutans to penicillin induces a 15-fold increase in phospholipid release from the organism, raised the question whether exposure of GBS to penicillin or to other beta-lactam antibiotics in newborns and mothers induces a release of GBS phospholipids causing pulmonary hypertension and respiratory distress in newborns. Analysis of a data file of the NICHD multicenter GBS study found a possible association between GBS colonization, penicillin treatment and respiratory distress in neonates: 8.8% of GBS colonized newborns who did not develop early-onset GBS disease had respiratory distress within 48 h after birth compared to 1-3% of non colonized newborns. Furthermore, colonized newborns of penicillin treated mothers were 2.62 times more likely to develop respiratory distress than those of GBS carrying but untreated mothers (95%CI:1.79-3.83). An assay to quantify cardiolipin in serum has been developed using HPLC-electrospray ionization mass spectrometry. It is being refined to measure cardiolipin levels in maternal and cord-blood samples. These levels will be related to GBS colonization, penicillin prophylaxis and neonatal respiratory distress.

A prospective study to relate serum GBS phospholipid levels to respiratory distress in newborns of GBS colonized mothers conducted at Baylor College of Medicine, Houston, Texas and Children's Hospital and Research Center Oakland, California was completed. Vaginal and rectal swabs were obtained during labor for GBS culture from mothers of at least 32 week gestation. A semiquantitative culture was used. Demographic and clinical data were obtained from medical records and the epidemiology of GBS colonization published. Three groups of GBS colonized mothers were assembled: untreated, beta-lactam antibiotic-treated, and treated with non-beta-lactam antibiotics. Maternal and cord sera from these groups were collected, bar-coded and stored at -70^oC for the cardiolipin assay. All newborns were monitored for signs of respiratory distress. Two groups of newborns were assembled: with and without respiratory distress, their serum samples collected, bar-coded and stored.

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

The potential of influenza viruses to develop into worldwide pandemics was demonstrated by the unchecked spread of the pandemic H1N1 2009 influenza A virus into populations lacking resistance to this novel strain. Although an estimate of up to 89 million Americans were infected, fortunately relatively few deaths (0.02%) occurred because of the low pathogenicity of this strain. However, the potential for a world health crisis with a severe influenza pandemic exists if a new influenza virus strain emerges with the typical high rate of infection seen with the H1N1 strain coupled with the unusually high 60% mortality rate of the H5N1 virus. Rapid vaccine production is needed. To this end we use recombinant DNA technology, cellular protein expression systems, and chemical conjugation methods to rapidly produce:

Hemagglutinin (HA). We have developed a method for rapid production of recombinant hemagglutinin (rHA) of influenza viruses. This procedure does not require access to virus strains. Using only DNA sequence data from the CDC/WHO influenza virus database, we synthesized chemically HA genes de novo, using a procedure optimized for protein expression in E. coli. Our protocol reduces the time required to produce an influenza vaccine to about one month. In addition we have developed a cloning protocol utilizing influenza virus RNAs as templates for reverse transcription-polymerase chain reaction (rPCR) gene amplification. Using these procedures, bacterial seed clones/cultures have been constructed and used to produce pilot plant quantities of rHA. We have now developed standardized downstream processing protocols for protein extraction, purification, refolding, and vaccine formulation. The rHA constructs were designed to represent the mature configuration of HA with the transmembrane domain, which spans the viral capsid, deleted from the carboxyl terminal. This domain was replaced with a Gly3X-His6X tag to facilitate purification of the expressed protein using Ni-ion column chromatography.

rHA from five representative strains, was produced in E. coli. The expressed protein was purified from inclusion bodies by urea solubilization and Ni-ion column chromatography. Solubilized rHA was further processed by rapid dilution into refolding buffer, extensive dialysis, and spin-filter concentration. Vaccine candidates were formulated by either adsorbing the rHA onto alum, formaldehyde treatment, or both. Injected into young outbred mice three times 2 weeks apart (2.5-5 mcg/mouse), rHA induced antibodies with hemagglutination inhibition titers of 40 or higher, suggesting that rHA could induce protective immune responses against influenza virus infection (FDA guidelines require a minimal titer of 40). Our data suggest that the alum-absorbed rHA vaccine, produced in just four weeks, can fulfill the FDA requirements.
M2e peptide conjugates. M2e is the 23-amino acid N-terminal polypeptide of the matrix 2 protein (M2) located on the capsid surface, and is highly conserved among influenza A strains. Recent studies showed that the exposed 23-amino acid M2e peptide genetically fused to the N-terminus of hepatitis B virus core particles conferred complete protection against a lethal heterologous influenza virus challenge in a mouse model.

A synthetic M2e peptide was linked to a genetically detoxified diphtheria toxin (rDT; H21G) via thioether linkages. MADLI-MS analyses showed an average of 7 chains of M2e per DT molecule with a mass ratio of DT:M2e = 1:0.3. This conjugate, in aqueous form or alum adsorbed, induced in mice statistically significant serum IgG responses after the 2nd and 3rd injections (0.4 to 1.4 p<0.0005). An antibody response was also observed against DT. Our preliminary results suggest that this candidate vaccine may induce immunity against heterologous strains of influenza A virus.
We used the HA fusion peptide (also a conserved region of influenza A viruses) of H5N1 virus for preparing investigational vaccines: two sequences were synthesized; 9 amino acids of the fusion peptide plus a linker, and 18 amino acids plus a linker were conjugated to BSA. Injected sc into young GP mice, at a fraction of a human dose, these conjugates were immunogenic and induced booster responses. The induced antibodies bound to rHA, the influenza virus, and to the peptides. This methodology of linking the highly conserved fusion peptide to carrier protein can broaden a protective immune response against influenza A virus strains and some influenza B strains. This would eliminate the need for annual vaccination.

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

Complete NMR characterization data have been measured for a series of fortimicin antibiotics, including the diamino-inositol component fortamine, fortimicins A, B, E, and isofortimicin. The data are comprised of 1D H-1 and C-13 NMR spectra, 1D H-1/N-15 HSQMBC, and 2D COSY, TOCSY, HSQC, and HMBC spectra. These data sets are under analysis. The 2D COSY and TOCSY are homonuclear H-1/H-1 data sets and are expected to confirm the H-1 NMR assignments. The 2D HSQC and HMBC spectra are heteronuclear H-1/C-13 data sets for confirmation of the C-13 assignments, while the 1D H-1/N-15 HSQMBC heteronuclear spectra contain information on the H-1/N-15 coupling constants. This project is currently dormant pending the availability of commercial software for the analysis of the anti-phase NMR spectra generated by the HSQMBC technique. The manual analysis procedure used previously has proved to be inadequate for this task, and various NMR software packages were found to be unsuitable. The subject of the stereochemical dependence of NMR coupling constants has been reviewed and published.

NMR verification of structures of bacterial saccharide precursors for vaccines

High-resolution nuclear magnetic resonance spectroscopy is a powerful method for analysis of the conformations, molecular structures, purity, and stereochemistry of saccharides of various lengths that are of prime interest in bacterial vaccine development. The general goals are determination, verification, or characterization of saccharide structures and purity by high-resolution NMR spectroscopy.

Work has continued on the NMR characterization of oligosaccharide intermediates of Vibrio cholera antigens, including saccharides up to the decamer level of complexity. The techniques used included high-resolution one-dimensional (1D) H-1 and 1D C-13 NMR at 500 MHz and 126 MHz, respectively. Validation of the chemical structures was based on integration of the 1D H-1 NMR spectra, and on counting of carbon resonances in the 13C spectra, especially in the well-separated spectral regions of the C=O, aromatic C, C-1, C-N, and C-CH3 moieties. The NMR spectra were also used to assess the purity of the synthetic preparations, for example, determination of the extent of completion of hydrogenolytic removal of benzyl protecting groups by measurement of the content of aromatic groups.

A comprehensive manuscript has been written on the organic synthesis of oligosaccharide components of the OSP of E. coli O148, including saccharides containing 4, 6, 7, 8, 10, 11, and 12 residues. Protein conjugates of these saccharides with bovine serum albumin and recombinant diphtheria toxoid have also been prepared, and their anti-LPS IgG immunochemical values measured and compared with those of the corresponding diphtheria toxoid conjugates of oligosaccharide components of the OSP of Shigella dysenteriae type 1 containing 8, 10, 11, 12, or 13 saccharide residues. The manuscript includes seven tables of data on the H-1 chemical shifts, C-13 chemical shifts, H-1/H-1 coupling constants, and 1J C-1,H-1 heteronuclear coupling constants of the 4-mer, 8-mer, and 12-mer 5-methoxycarbonylpentyl glycosides, and a two-part figure indicating the similarity of the 1H-coupled HSQC NMR spectra of the 8-mer and 12-mer glycosides, but also how these oligomers may be distinguished by the intensities of the cross-peaks in the spectra.

In the course of validating the structures of synthetic BBGL-2 variants by 1D and 2D NMR methods, we have now made detailed H-1 and C-13 NMR assignments for four of these structures; namely, 1,2-di-O-oleoyl-, 1-O-oleoyl-2-O-palmitoyl-, 1-O-palmitoyl-2-O-oleoyl-, and 1,2-O-palmitoyl-3-O-alpha-D-galactopyranosyl-sn-glycerols. Interpretation of the C-13=O chemical shifts of the four galactosyl diglycerides has led to a set of rules that describe the dependence of these C-13 shifts on fatty acid type, and substituent position on the glycerol moiety. The rules are useful for NMR analysis of the mixtures of products that result from acylation reactions of imperfect regiospecificity. The NMR data for the four diglyceride analogs have been published as part of a detailed report on the synthesis and antigenicity of BBGL-2 analogs.

A Polysaccharide Vaccine for Mycobacterium tuberculosis

Tuberculosis remains a serious and common disease worldwide. Worldwide emergence of multi-antibiotic resistant strains, accelerated by the passage of M. tuberculosis in patients with AIDS, poses a public health problem. Although it is the most frequently used vaccine in the world, there is no scientifically-based evidence that BCG prevents primary pulmonary tuberculosis (the most common disease caused by M. tuberculosis). The protective effect of BCG vaccination against meningitis in children has not been related to an antigen/s or a host immune component. Research into a new vaccine is based upon the similarity of primary infection caused by M. tuberculosis with that of capsulated bacterial respiratory pathogens, viz: 1) tuberculous meningitis has a similar age distribution as meningitis caused by meningococci, pneumococci and Haemophilus influenzae type b; 2) M. tuberculosis and other mycobacteria have a polysaccharide capsule in vitro and in vivo; and 3) Antibodies to protein components prolonged survival of but do not protect animals against challenge with wild-type M. tuberculosis. We purified a glucan and an arabinomannan from a saline extract of M. tuberculosis. The polysaccharides were conjugated to P. aeruginosa recombinant protein A and shown to be immunogenic in mice and in rabbits. Antibodies induced by these conjugates, whether actively induced or passively administered, did not protect mice against pulmonary challenge with M. tuberculosis. Adult patients with tuberculosis had higher levels of antibodies (ELISA) to both polysaccharides than normals. A standardized and quantitative assay for diagnosis of tuberculosis with these polysaccharide antigens is underway. Both the glucan and arabinomannan conjugates are being evaluated for their protective activity in mice in collaboration with CBER, FDA.

Publications

Passwell JH, Ashkenazi S, Banet-Levi Y, Ramon-Saraf R, Farzam N, Lerner-Geva L, Even-Nir H, Yerushalmi B, Chu C, Shiloach J, Robbins JB, Schneerson r, The Israeli Shigella Study Group. Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1-4-year-old Israeli children. Vaccine 2010;28:2231-5.
Kubler-Kielb J, Vinogradov E, Lagergård T, Ginzberg A, King JD, Preston A, Maskell DJ, Pozsgay V, Keith JM, Robbins JB, Schneerson R. Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine. Proc Natl Acad Sci USA. 2011;108:4087-92.
Lin FY, Weisman LE, Azimi P, Young AE, Chang K, Cielo M, Moyer P, Troendle JF, Schneerson R, Robbins JB. Assessment of Intrapartum Antibiotic Prophylaxis for the Prevention of Early-onset Group B Streptococcal Disease. Pediatr Infect Dis J 2011;30:759-63.
Chen Z, Schneerson R, Lovchik J, Lyons CR, Zhao H, Dai Z, Kubler-Kielb J, Leppla SH, Purcell RH. Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to Bacillus anthracis capsule. Proc Natl Acad Sci USA. 2011;108:739-44.
Pozsgay V, Kubler-Kielb J, Coxon B, Marques A, Robbins JB, Schneerson R. Synthesis and antigenicity of BBGL-2 glycolipids of Borrelia burgdorferi, the causative agent of Lyme disease. Carbohydr Res. 2011;346:1551-63.

Collaborators

Shai Ashkenazi, MD, Schneider Children's Hospital, Tel Hashomer, Israel
Parvin Azimi, MD, Children's Hospital and Research Center at Oakland, Oakland, CA
Peter Backlund, PhD, Section on Mass Spectrometry & Metabolism, NICHD
Joseph Bellanti, MD, Immunology Center, Georgetown University Medical Center, Washington, DC
Zhaochun Chen, PhD, Laboratory of Infectious Diseases, NIAID
John Clemens, MD, International Vaccine Institute, Seoul, Korea
John Clements, PhD, Tulane University School of Medicine, New Orleans, LA
Jerri Curtis, MD, Laboratory of Biochemistry, NHLBI, Bethesda, MD
Kim Y. Green, PhD, Epidemiology Service, NIAID
Yasutaka Hoshino, DVM, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Albert Z. Kapikian, MD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Jerry King, PhD, Dept. of Molecular & Cellular Biology, University of Guelph, Ontario, Canada
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
Richard Malley, MD, Children's Hospital, Tufts University School of Medicine, Boston, MA
Laura O. Martin, PhD, Novartis Institute for Global Health, Siena, Italy
Louis Miller, MD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
Mark A. Miller, MD, Fogarty International Center, NIH, Bethesda, MD
Robert H. Purcell, MD, Laboratory of Infectious Diseases, NIAID
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
Yimin Wu, PhD, Lab of Malaria Immunology & Vaccinology, NIAID
Guilin Xie, PhD, Lanzhou Institute of Biological Products, Lanzhou, Gan-Su, China
Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

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