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Development of Vaccines Against Diseases Especially in Children

  • John B. Robbins, MD, Joint Program Director, Program in Developmental and Molecular Immunity
  • Rachel Schneerson, MD, Joint Program Director, Program in Developmental and Molecular Immunity; Head, Section on Bacterial Disease Pathogenesis and Immunity
  • Audrey L. Stone, PhD, Senior Investigator
  • Genene Beyene, BScEng, Charles River Research Assistant
  • Zuzanna Biesova, PhD, Postdoctoral Fellow
  • Chi-Chang Chen, PhD, Research Fellow
  • Faye C. Chen, MS, Technician
  • Chiayung Chu, MD, Staff Scientist
  • Bruce Coxon, PhD, DSc, Senior Research Fellow
  • Zhongdong Dai, MD, Adjunct Investigator
  • C. Goran Ekborg, PhD, Oak Ridge Fellow
  • Peter Ftacek, PhD, Research Fellow
  • Ariel Ginzberg, PhD, Postdoctoral Fellow
  • Chunyan Guo, BSc, Charles River Research Assistant
  • Steven W. Hunt, BSc, Research Assistant
  • Arthur B. Karpas, PhD, Charles River Adjunct Investigator
  • Galina Kazanina, PhD, Senior Research Assistant
  • Jerry Keith, PhD, Oak Ridge Fellow
  • Joanna Kubler-Kielb, PhD, Research Fellow
  • 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
  • Christopher P. Mocca, MS, 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
  • Alaa Shaban, BS, Intramural Training Assistant
  • 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. For example, in Brazil, the incidence of Hib meningitis decreased by 69 percent during the first year after initiation of Hib conjugate immunization while the incidence of Hia meningitis increased 8-fold. The Netherlands Reference Laboratory reported that type a was observed only in children younger than 4 years old. 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 either 8 or 12 repeat units, with the terminal keto groups used for conjugation to aminooxylated bovine serum albumin (BSA) or to tetanus toxoid. We injected the conjugates into mice, three times at 2-week intervals at 2.5 mcg/mouse and obtained sera a week later. ELISA demonstrated antibodies to both Hia and Hib, with the octamer conjugate a better immunogen than the dodecamer conjugate. Some of the tested sera showed bactericidal activity against both type a and type b as correlated roughly with their ELISA values. Other constructs and formulations are being studied. We are also synthesizing the repeat unit of Hia CP; glycosil-ribitol-phosphate. A dimer has been prepared thus far.

Bordetellae and Haemophilus ducreyi

The licensed pertussis vaccines confer incomplete efficacy on a personal basis, probably because pertussis toxin antibodies do not kill the organism directly, however herd immunity contributes to almost complete protection with wide vaccine usage. Induction of bactericidal antibodies would increase vaccine effectiveness on an individual basis. 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 of their protein conjugates.

Both 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, significantly higher than those induced by a conjugate of B. bronchiseptica core + full O-SP. Mutants deficient in O-SP were 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 the three non-reducing end core saccharides repeated several times. 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.

Shigellae

The O-SP of Shigella sonnei bound to recombinant non-toxic P. aeruginosa exoprotein A had an efficacy of over 70 percent in young adults exposed to 6 to 14 percent attack rates. This and a S. flexneri 2a conjugate evaluated in a Phase 3 study in 1- to 4-year-olds, with each conjugate serving as a control for the other, showed the vaccines to be safe. Immunogenicity and efficacy of the S. sonnei conjugate were age-related with 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 using the model of synthetic S. dysenteriae type 1 saccharides-protein conjugates. 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. The applicability of this approach to S. flexneri types 2a and 6 and S. dysenteriae type 1 is being investigated.

Peptide-protein conjugate vaccines

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

Peptides of D-gamma-glutamic acid (D-G-PGA) of the capsule of B. anthracis 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) for preparing humanized monoclonal antibodies. 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 postexposure therapy for anthrax.

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

  1. 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. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased further the antibody levels. Similar results were obtained with Pvs25-Pvs25 conjugates. Transmission blocking activity of immune Pfs25- Pfs25 sera correlated with antibody levels measured by ELISA.
  2. Following earlier studies using NANP (Asn-Ala-Asn-Pro), the repeat fragment of the pre-erythrocytic parasite stage as a vaccine, and following knowledge we gained from S. dysenteriae type1 synthetic O-SP oligosaccharide and from B. anthracis capsule derived peptide- protein conjugates, 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, with a terminal Asn as NANP or NPNA, being 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.

We then combined the two approaches, binding NANP repeats to Pfs25-Pfs25. This experimental vaccine induced in mice antibodies to both its components, with secondary biological activities of transmission blocking and binding to sporozoites in IFA. The Pfs25-Pfs25 imparted its long lasting antibody properties to NANP. Pfs25 by itself was not immunogenic nor a carrier. The NANP-Pfs25-Pfs-25 conjugates would provide both individual and community based protection.

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 non toxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA). An efficacy of 89% at 47 months was shown in 2- to-5-year olds injected with Vi-rEPA. 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 evaluated in 301 Vietnamese infants. Controls received Hib-TT +EPI or EPI alone. 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.

Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries, transmitted through ingestion of food or drink contaminated by infected persons. A chronic carrier was identified in an investigation of SPA outbreak by measuring serum anti-LPS levels among SPA patients. An expanded survey for the identification of SPA carriers was initiated in a high endemic area of China in collaboration with the Guangxi Center for Disease Prevention and Control.

Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that serum vibriocidal activity is directed toward its LPS. In a Phase 1 trial, V. cholera O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. A hexamer corresponding to the O-SP was chemically synthesized and conjugated to TT. In mice this conjugate elicited higher vibriocidal activities than the native O-SP conjugate used in the Phase I study. Various linker lengths were studied. A conjugate synthesized with a heptadecamer linker was more immunogenic than the one with a nonamer linker.

Rotavirus is the most common cause of infantile diarrhea worldwide. Two licensed oral rotavirus vaccines confer limited protection and some lots were found to contain small amounts of porcine circovirus 1 and 2 DNA. We are designing a parenteral vaccine based on capsid proteins. 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 polysaccharide vaccines improved protein solubility. The core region of the capsid protein is also 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, and hemolytic uremic syndrome (HUS). HUS is a major cause of acute and chronic kidney damage in young children and could 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

Shigella dysenteriae type 1 causes the most severe forms of shigellosis, both endemic and epidemic. No shigella vaccine is licensed. To prepare an LPS-based vaccine, the repeat unit of the O-specific polysaccharide of dysenteriae type1 was synthesized: 3)-alpha-Rhap-(1,2)-alpha-GALp-(1,3)-alpha-GLCNACp-(1,3)-Rhap-(1. BSA conjugates of the synthetic octa- to hexadeca-saccharide fragments of the O-SP elicited specific IgG antibodies in mice. The highest antibody levels were obtained with the deca- and undecasaccharide-BSA conjugates where the non-reducing terminal residue was either N-acetyl-glucosamine or galactose. Clinical lots of the 2 oligosaccharides bound to a genetically inactivated diphtheria toxin were prepared using oxime conjugation.

E. coli O148. It has been proposed that S. dysenteriae type 1 gained its O-SP from E. coli O148, and shares the same O-SP repeat unit, except that the glucose residue in the E.coli O148 O-SP is replaced by a galactose residue in the S. dysenteriae type 1; both residues having identical alpha anomeric configurations. To analyze the cross-reactivity between these two bacteria, we have synthesized a panel of E. coli O148-related oligosaccharides and their protein conjugates, using the oxime technology. Cross-reactivity was established by the following findings: 1. Antisera raised by either (killed) E.coli O148 bacteria or by its synthetic dodecasaccharide-BSA conjugate, bound to the lipopolysaccharides (LPS) of both organisms by immunoblotting. 2. Similarly, antisera raised by killed S. dysenteriae type 1 or by the synthetic type 1 dodecasaccharide-BSA conjugate, bound to both LPSs. 3. The O-SPs of both bacteria inhibited the binding of homologous and heterologous oligosaccharide-protein conjugates induced sera. Based on these findings, we hypothesize that a saccharide-based vaccine against one of these organisms will offer protection against both.

Borrelia burgdorferi causes Lyme disease afflicting a large number of people of all ages, including children, in large areas of the US, prominently in the northwestern part of the country. A protein-based vaccine has been withdrawn by the manufacturer.

The bacterium expresses two major glycolipids on its surface: BBGL-1 and BBGL-2. We have chemically synthesized BBGL-1 and its protein conjugate: both elicited BBGL-1 antibodies in mice. BBGL-2 contains alpha-galactosyl-glycerol, with acyl groups of various chain lengths at two hydroxyls (HO-1 and 2) of the glycerol moiety, e. g. palmitoyl, oleoyl moieties. However, the exact locations of the individual fatty acids are not known. To map the probable structure of the individual components we synthesized a number of potential BBGL-2 congeners and tested their ability to bind to B. burgdorferi antiserum in a dot-blot assay: preparations that contained palmitoyl groups at both hydroxyls did not bind, independently of the anomeric configuration of the galactose residue or of the configuration of the glycerol moiety. Neither did the one that had a palmitoyl group at the HO-1 position with the H-2 position nonacylated. On the other hand structures that contained two oleolyl groups bound to the B. burgdorferi antiserum, again, independently of the anomeric configuration of the galactose. These and other findings establish lead structures for the development of a vaccine against B. burgdorferi.

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. Curtis et al. purified and identified cardiolipin and phosphatidylglycerol from the GBS cell wall as pulmonary hypertensive compounds. Others have demonstrated that exposure of Streptococcus mutans to penicillin induces a 15-fold increase in phospholipid release from the organism. These findings 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 in Dr. Alfred Yergey's laboratory using HPLC-electrospray ionization mass spectrometry. This assay is being refined to measure cardiolipin levels in blinded maternal and cord-blood serum 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 was conducted at Baylor College of Medicine, Houston, Texas and Children's Hospital and Research Center Oakland, California. Data analysis is underway. Vaginal and rectal swabs were obtained during labor for culture from mothers of at least 32 week gestation. A semiquantitative culture was used to determine their degree of colonization. Demographic and clinical data were obtained from medical records. 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 last year 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 pandemicexists 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.

A rapid response to an influenza pandemic is impossible because the egg-based vaccine manufacturing methods cannot produce enough vaccine in the required time. It takes 9 months for vaccine manufacturers to produce the vaccine, requiring 1 to 2 eggs per vaccine dose. This method, with a maximal capacity of 300 million doses, is inadequate to produce the 2-3 billion vaccine doses required to prevent a worldwide pandemic.

Our approach to this challenge is to use recombinant DNA technology, cellular protein expression systems, and chemical conjugation methods to rapidly produce:

  1. Hemagglutinin (HA). This protein forms homotrimer-spikes on the surface of the virus capsid. FDA mandates that all influenza vaccines have at least 15 mcg of this immunogen per dose. 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 chelating 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 preliminary data suggest that the alum-absorbed rHA vaccine, produced in just four weeks, can fulfill the FDA requirements. Antibody neutralization experiments based on plaque reduction analysis and inhibition of viral replication within a unique tissue culture cell line are planned.

  2. M2e peptide conjugates. M2e is the 23-amino acid N-terminal polypeptide of the matrix 2 protein (M2) located on the capsid surface. Unlike the virion surface proteins HA and NA, which are subject to constant genetic drift and shift, M2 is highly conserved in all influenza A strains likely due to its protected location within the viral membrane, preventing a host immune response; it has therefore the potential of broadening the protective immune response against 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.

    We linked covalently a synthetic M2e peptide 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, was injected s.c. into mice at 2.5 mcg peptide in 0.1 mL PBS 2 or 3 times, 2 weeks apart. Sera were collected 1 week after the last injection. Both the M2e-rDT and the alum absorbed M2e-rDT conjugates induced statistically significant serum IgG responses after the 2nd and 3rd injection (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.

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

The goals of studies in the heparin glycobiochemistry lab include research on which multi-functional heparin/heparan sulfate biopolymers (H/HS) govern healthy growth in young children and the maintenance of well-being thereafter. The goal is to elucidate the roles of H/HS in normal and disease processes, and to develop drugs which prevent or treat diseases caused by pathogens known to be inhibited by heparins, such as HIV-1 and malaria, through preclinical and clinical studies.

Our first aim is to reveal and develop a family (macro combinatorial library) of heparin-mimetic S-OligoS and then to establish structure-function relations of individual family Components and identify those that act, as do heparins, specifically against given pathogenic infections, and might help to devise means to identify protective antigens. The second aim is to complete ongoing preparation of a novel anti-AIDS drug (SOLIS) for clinical studies from a library Component (PkII, CpF) which is a potent in vitro inhibitor of HIV-1 fusion reaction(s) based on inhibition of syncytium-formation assays.

Consistent with our findings that SOLIS is compatible for combined formulation, another aim is to elucidate the compatibilities and inhibitory effects of co-treatment, especially the relevance to combating the unexplained 8 percent increase in incidence of HIV infection predicted to occur in malaria endemic regions. Physical, chemical and optical techniques are applied, modified and/or devised aimed at elucidating primary structures and conformations of the S-oligoS to elucidate their functional heparin-mimetic sulfate and carboxyl geometries. Equipment and biological regents are devised and prepared, including those aimed at automation and clinical markers.

HIV-infection in Americans is one million plus and rising, 50 times that worldwide. With no cure or effective vaccine, and the successful, long term HAART control of viral load and mortality in developed countries threatened by toxicity, multi-drug resistant strains, and reliance on mostly protease and reverse transcriptase inhibitors, effective inhibitors against viral components having other functions in HIV-1 attack are critically needed. Heparin has been shown to inhibit binding and fusion of AIDS virus to its target CD4 cell in vitro, but heparin is an antithrombin-activator anticoagulant and as such may not be useful clinically because of possible hemorrhagic toxicity. H/HS are highly sulfated, found in various tissues and comprise modulator/ receptor systems throughout the body. They not only govern the level of activity/function of numerous normal proteins and cell systems, but facilitate the infectiousness of various human pathogens in unclear mechanism(s). H/HS have a large diversity of sugar modifications and sulfate density (anionic density) along their polysaccharide chain. This structural diversity provides H/HS with a sufficiently large number of unique sequences to account for its known ability to modulate uniquely the functions of many diverse normal and disease processes. e.g., development, growth, inflamation, and attack by viruses and other human pathogens. Due to the high degree of similarity of physicochemical properties of H/HS chains carrying these sequences, and lacking knowledge of their given specific ligands with which to achieve their isolation, libraries of the unique H/HS have not been obtainable for research. The lack of the purified ligand in most H/HS functional reactions blocks progress and is of increasing concern.

We discovered that heparin, thought to be a random polymer lacking secondary structure/ information content, assumed a helical structure in solution. We classified the various sulfated glycosaminoglycan biopolymers (GAG, mucopolysaccharides) based on specificity of their optically active structures, and demonstrated (AL Stone 1963/4 Biopolymers 2/3) that H/HS (and keratosulfate) comprise a Class of GAGs, separate from other GAG types. We also studied the macrostructure of numerous other highly sulfated natural polysaccharides and developed means for their qualitative and quantitative measurement. These methods and a German heparin-mimetic pharmaceutical (chemically sulfated hardwood xylan) which mimicked unfractionated heparin at similar uM concentrations in almost all its known biological actions, were used early to develop a pilot macro combinatorial library of sulfated oligosaccharides based on the structure-function model for the antithrombin-dependent anticoagulant heparin (R.D. Rosenberg, et al). This library demonstrated for the first time that in vitro inhibitions of HIV-1 cytotpathology and syncytium-formation were 1) each governed by a degree of structural specificity and 2) each were separable from the anticoagulant S-oligoS of the pharmaceutical and from each other, two essential indicators of their potential usefulness for further drug development (AL Stone, et al 1998 Glycoconjugate Journal 15:697-71).

S-OligoS Library

We are currently purifying three S-OligoS: one of minimum mass required to retain full capacity to inhibit the cytopathic effects of HIV and two of adjacent masses, one smaller, one larger than CpC: for elucidation of structure-function relation, e.g., alternative sugar ring conformations, mass, oligoS conformation(s); to prepare derivatives (conjugates), e.g.,with bifunctional fluorescent probes, with select polypeptides (conjugate antigens).

SOLIS: A Potential HIV-1 Fusion Inhibitor:

Orally administered S-oligoS are very poorly absorbed, necessitating an i.v. route of administration and consequently a relatively large amount of drug. The first batch of product has been completed and is stored at -75 degrees C. Currently, a second award of 40 grams of the formulated pharmaceutical is under study. The original pharmaceutical is now processed to remove a weak amber color before it is formulated (FDA required). A comparison of fractionation processes to indicate any needed adjustments due to this treatment showed only minor, adjustable shifts in the chromatogram.

Studies on Combination Therapy:

The results of co-treatment in the HIV-1 cytopathology protection test need to be amplified but suggest that the interdependence of disease susceptablity between HIV and malaria may not reside in cell membrane reactions.

Virus Ligands From Bifunctional SOLIS and Other S-OligoS Probes:

We propose to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection by using a bifunctional SOLIS probe to achieve chemical bonding to endogenous viral/cell proteins during virus attack. Subsequent isolation of the putative endogenous protein-probe will continue and include further purified CpC. Such ligand(s) could be used to obtain endogenous receptors and/or might be protective antigens.

Structure-Function Relations:

S-oligoS remaining after chemical sulfation of native xylan comprise a family of oligomers of about 20 to less than 2 K average mass. Compositional and structural analysis of Cps indicate a tetraS motif within the S-OligoS chains: D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motifs. We also found varying axial sulfates (alternative chair conformations) which are displayed in decreasing proportions with decreasing mass. Currently, CpC region S-6ligoS are ready for advanced FTIR spectral analysis and HMQC correlation spectra to determine the relation of alternative chair structure in the S-OligoS and the inhibition of HIV-1 gp120 and gp41, and in anticoagulation. Details of such structures must be confirmed before small molecule-substitutes might be considered for drug development.

Heparin-mimetic sulfated oligoxylan inhibitors of malaria parasites

The goal of the heparin glycobiochemistry lab includes research on the role of heparin/heparin sulfate biopolymers (H/HS) in normal and disease processes, which will enable development of drugs that prevent or treat diseases known to be inhibited by heparins, such as HIV-AIDS and malaria.

This includes the expansion and new applications of our macromolecular combinatorial family of H/HS mimetic sulfated oligoxylans (S-OligoS) in its potential anti-malaria parasite components. Objectives are: to improve preparative methods and obtain larger quantities of the specific S-OligoS which we discovered is a potent inhibitor of the primary invasion of hepatocytes by the parasite in vitro; to continue structural elucidation which provides knowledge about specificity and the endogenous ligand(s), as well as usefulness in vaccine design, and which may lead to smaller S-OligoS suitable for high throughput techniques in antimalarial drug development.

An additional goal is to identify component(s) of our H/HS family that ameliorate or prevent cerebral malaria in young children. S-OligoS would be tested for inhibitory capacity in an vitro erythrocyte rosetting assay. Another goal addresses the increased incidence and spread of HIV infection in malaria endemic regions, due to a mutual enhancement of the vulnerability of either patient to the other pathogen. A first objective was to develop an assay to elucidate effects of combination dosage of our two pathogen-selective S-OligoS against HIV.

Severe and cerebral malaria kill about 2-3 million people per year; 90 % are young children. There is no preventive vaccine, and the malaria parasites are increasingly resistant to anti-malarial drugs. The initial infective process is between the invading parasite and hepatocytes of the host. In several studies, the heparin/heparin sulfate (H/HS) receptor systems have been implicated in this invasion. We previously applied our combinatorial family of heparin-mimetic S-oligoS to characterize their in vitro inhibition against malaria parasites in vitro and revealed that the heparin-based inhibition of P. yoelii sporozoite invasion of hepatocytes was governed by structural specificity and was concentration dependent. Two highly active library components (Cp) were identified, Cp6 and Cp11, with estimated EC 50s of 3.5 and 5 micromolar, respectively; Cp11 has displayed negligible heparin-anticoagulant capacity against thrombin.

Inhibitory Capacity

Further studies of the inhibitory capacities of Cp11 and Cp6 against liver stage development of malaria parasites in vitro are planned. Our renewed mutual Confidentiality Agreement with Bene Arzneimittel, Munich, D.R. has provided another large lot of heparin-mimetic pharmaceutical material for preparation of additional Cp11 and our HIV-1 inhibitors, SOLIS and the Cp 8 group. Development of an enlarged preparation of Cp11 is ongoing.

Structural Study

Spectroscopic and chemical analyses showed that the H/HS-mimetic S-OligoS Cps carries sulfate groups on up to 85 percent of the sugar hydroxyls (high anionic density) and contain a specific tetrasaccharide motif of three 1-4 beta linked xyloses and a 1-2 alpha-linked glucuronic acid, one third or less O methylated (as a branch on the xylan chain). In addition, the number of these alternate forms appears to increase with increase in mass of the Cp, and induces specific distance and angle alterations in the otherwise uniform S-OligoS chains. Such a motif could accommodate the variations in geometries involving sulfates that are required for multifunctional mimicry of the heparins. Further elucidation of the Cp11 structure might enable application of advanced high throughput methods in further drug development. Cp11 and Cp6 samples are in progress for FTIR and NMR analyses.

Cerebral Malaria

We anticipate testing our H/HS-mimetic combinatorial family against the acute cerebral distress and fatality in patients with cerebral malaria as soon as collaboration with a malarial parasitologist is arranged.

"Copathogenesis"

Recently calculations (Science 319:1603 2006) show that HIV infection in malaria endemic regions is spread at a rate 8 percent higher than in other regions where HIV-AIDS is on the rise. We suggested previously that co-treatment of malaria and HIV-AIDS would be feasible because of the close similarity in the physicochemical properties of the respective S-oligoS inhibitors. Our preliminary in vitro study on combined dosing of Cp11 and SOLIS (S-OligoS malaria parasite and HIV-1 inhibitors, resp.) confirmed the compatibility of the two potential drugs in assays using CM-SS cells. The data will be used to generate appropriately designed assays which will be used with the Cp11 to elucidate this "copathogenesis".

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

To define the stereochemical dependence of nitrogen-15-proton coupling constants in amino sugars by high-resolution NMR measurements of these coupling constants in model compounds. For room temperature NMR probes of the Bruker TXI or BBO type, we used the Carr-Purcell-Meiboom-Gill heteronuclear single quantum multiple bond correlation (CPMG-HSQMBC) NMR procedure. Based on data measured for amino sugar derivatives during the past two years, we expanded our objective from definition of the dihedral angular dependence of the coupling constants to include the general stereochemical dependence of these couplings, atomic electronegativities and their orientation. We are working on application of the nitrogen-15 NMR data to determine or verify the structures of nitrogen-containing bacterial polysaccharides, and aminoglycoside antibiotics from the fortimicin group.

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.

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.

During the year, a further 17 monosaccharide and oligosaccharide intermediates of Vibrio cholera antigens, for the preparation of a hexasaccharide-protein conjugate have been analyzed by high-resolution one-dimensional (1D) H-1 and 1D C-13 NMR at 500 MHz and 126 MHz, respectively, making a cumulative total of 80. 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.

H-1 NMR spectra at 500 MHz have been measured for the characterization of 12 native polysaccharide fractions isolated from Shigella sonnei. Comparison of the integrals of the polysaccharide H-1 and rhamnose methyl signals facilitated detection and quantitation of core polysaccharide in the samples. The techniques provide complex fingerprint spectra that can be compared with literature data to verify identity. A sample of the E. coli O148 O-specific polysaccharide has been characterized by H-1 NMR.

When we came to publish our work on immunochemical studies of Shigella flexneri 2a and 6, etc, a referee of the manuscript was concerned that acetyl groups on the polysaccharide might react with our conjugation-assisting reagent O-(3-thiopropyl)hydroxylamine, under the conditions of the conjugation reaction. We therefore conducted a study in which the O-SP of S. flexneri 6 was treated with an equimolar proportion of the hydroxylamine derivative under these conditions. The solution in D2O was monitored by H-1 NMR spectroscopy at various times over a period of 22 h. No changes in the relative intensities of the OAc and NAc resonances of the polysaccharide or in any other parts of the spectra were observed that would indicate an on-going chemical reaction.

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

One-dimensional and two-dimensional high-resolution NMR spectra have been acquired at 500 MHz from synthetic oligosaccharide fragments of the O-specific polysaccharide (OSP) of E. coli O-148, which cross-reacts with the S. dysenteriae type 1. The repeating units (RU) of the OSPs of these bacteria are closely related, the difference being the presence of D-glucose in the E. coli OSP RUs instead of D-galactose in the S. dysenteriae type 1. Other recent work (Feng et al.) has demonstrated that the OSP of E. coli O148 is an evolutionary precursor of S. dysenteriae type 1, and that the ability of the latter organism to incorporate D-galactose in its OSP has been acquired by plasmid exchange of a gene for a galactosyl transferase. The oligosaccharides studied include one, two, or three RUs of the OSP of E. coli O148, derivatized as their 5-methoxycarbonylpentyl glycosides, in preparation for coupling to a carrier protein to prepare a protein-saccharide conjugate.

Borrelia burgdorferi

One of our research programs involves the synthesis of glycolipid antigens for Borrelia burgdorferi (Lyme disease), and the preparation of their protein conjugates. Our initial publication described the isolation and characterization of two types of antigens expressed on the surface of the bacterium; cholesteryl 6-O-acyl-beta-D-galactopyranosides (BBGL-1) and 1,2-di-O-acyl-3-O-alpha-D-galactopyranosyl-sn-glycerols (BBGL-2). BBGL-1 analogs have been synthesized in our laboratory, and compounds of the BBGL-2 type in several labs, including ours, but usually without detailed NMR assignments. Kinjo et al. have demonstrated that the BBGL-2 analogs stimulate natural killer T-cell hybridomas to produce the anti-inflammatory cytokine interleukin-2, a property that has been used to assess the apparent antigenicity of the analogs. Such antigenicity was found to depend significantly on acyl-chain length and degree of saturation.

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;e-pub:ahead of print.
  • Robbins JB, Kubler-Kielb J, Vinogradov E, Mocca C, Pozsgay V, Shiloach J, Schneerson R. Syntheses, characterization and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. PNAS 2009;106:7974-7978.
  • Kubler-Kielb J, Majadly F, Biesova Z, Mocca CP, Guo C, Nussenzweig R, Nussenzweig V, Mishra S, Wu Y, Miller LH, Keith JM, Liu TY, Robbins JB, Schneerson R. A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates. Proc. Nat'l. Acad. Sci USA 2010;107:1172-7.
  • Szu S, Robbins JB, Schneerson R, Lin F-Y. Safety and Immunogenicity of Vi conjugate vaccines for typhoid fever in adults, teenagers, and 2- to 4-year-old children in Vietnam. In New Generation of Vaccines, 4th Ed. Levine MM, Kaper JB, Rappuoli R, Liu MA, Good MF, eds. Marcel Dekker, Inc. (NY, NY) 2009;489-96.
  • Biesova Z, Miller MA, Schneerson R, Shiloach J, Green KY, Robbins JB, Keith JM. Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus. Vaccine 2009;27:6234-8.

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
  • George Carlone, PhD, Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, GA
  • 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
  • Marc S. Lewis, PhD, Intramural Research Program, NIBIB, Bethesda, MD
  • Maria O. Longas, PhD, Purdue University, Hammond, IN
  • Richard Malley, MD, Children's Hospital, Tufts University School of Medicine, Boston, MA
  • James McMahon, PhD, Molecular Targets Development Program, NCI, Frederick, MD
  • Will Milhous, PhD, Div. of Experimental Therapeutics, Walter Reed Army Institute of Research, Forest Glen, MD
  • Louis Miller, MD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
  • Mark A. Miller, MD, Fogarty International Center, NIH, Bethesda, MD
  • Patrick R. Murray, PhD, Department of Laboratory Medicine, Clinical 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
  • John Sacci, Jr., PhD, Microbiology & Immunology, University of Maryland, College Park, MD
  • Allan Saul, PhD, Novartis Institute for Global Health, Siena, Italy
  • 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
  • Richard J. Webby, PhD, Saint Jude Children's Research Hospital, Memphis, TN
  • Leonard E. Weisman, MD, Baylor College of Medicine, Houston, TX
  • Xiaobo Wen, PhD, Laboratory of Infectious Diseases, NIAID
  • Yimin Wu, PhD, Lab of Malaria Immunology & Vaccinology, NIAID
  • Guilin Xie, PhD, Lanzhou Institute of Biological Products, Lanzhou, Gan-Su, China
  • Jin Yang, PhD, Guangxi Center for Disease Prevention and Control, Naning, Guangxi, China
  • Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

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