John B. Robbins, MD, Chief, Laboratory of Developmental and Molecular Immunity
Audrey L. Stone, PhD, Senior Investigator
Kimi F. Lin, MD, MPH, Medical Officer
Galina Kazanina, PhD, Senior Research Assistant
Jerry M Keith, PhD, ORISE ¹ Fellow
Zuzana Biesova, PhD, Postdoctoral Fellow
Faye Chingfei Chen, MSc, Technician
Elizabeth Ogbonna, BSc, BA, Charles River Research Assistant

In the form of capsule or lipopolysaccharide, surface polysaccharides of Gram-negative enteric pathogens function as both essential virulence factors and protective antigens. Serum IgG antibodies to these surface polysaccharides confer immunity by killing or inactivating the inoculum of the pathogen. We enhanced the immunogenicity of polysaccharides, oligosaccharides, and peptides by binding them to carrier proteins.

Molecular design, synthesis, and testing of recombinant protein immunogens for influenza vaccines development

Keith, Biesova; in collaboration with Miller, Shiloach

The causative agent for human influenza or respiratory flu disease is Orthomyxovirus. This enveloped RNA virus from the Orthomyxoviridae family has a single strand, negative sense, segmented genome. Influenza-associated complications cause the death of an estimated half a million people every year, including 36,000 deaths in the United States, primarily in the very young and elderly populations. During the 1918-1920 "Spanish flu" pandemic, influenza killed 20 to 40 million people worldwide. Influenza virus thus imposes a serious burden of disease. A highly mobile world population coupled with the threat of a human influenza pandemic emerging from the highly pathogenic avian H5N1 strain of influenza A virus has world health officials rethinking strategies for the best preventive approach should a pandemic become reality. In modern medical history, we have no precedent to guide the difficult scientific decisions necessary to protect the entire world population against such a formidable pathogen. Current influenza vaccines are produced in embryonated chicken eggs, a manufacturing protocol developed over a half-century ago. In a world emergency, industrial surge capacity for the production of vaccines made in this manner will not be able to meet vaccine demand in time to stop the spread of a highly pathogenic influenza virus.

By focusing on the primary protective immunogen of influenza vaccines, we have developed a protocol to produce an effective vaccine against any new strain of influenza virus within three to four weeks of receiving the virus strain and the nucleotide sequence of the gene encoding the hemagglutinin protein. As a demonstration of such a protocol, we have produced a modified recombinant hemagglutinin (rHA) protein for use as a candidate vaccine against the highly pathogenic H5N1 avian influenza A/Vietnam/1203/2004 virus. Using molecular biology techniques, we cloned the gene encoding the HA protein from native influenza virus isolated from an individual who died of the viral infection; we then produced the rHA protein in E. coli. We formulated purified rHA protein antigen into several vaccine candidates by using various combinations of aluminum hydroxide (AlOH3) and formaldehyde. The vaccine candidates are now being tested in mice for their ability to produce anti-rHA antibody and for the ability of the immune sera to inhibit hemagglutinin (HAI) production of red blood cells (RBCs) caused by the H5N1 influenza virus. We will use a mini-neutralization assay with MDCK cell culture to determine if the mouse immune sera can prevent replication of H5N1 influenza virus in cell culture. Preliminary test results are extremely encouraging.

Neonatal respiratory distress related to colonization with group B streptococci

Lin, Ogbonna; in collaboration with Yergey

About 25 percent of women of childbearing age carry group B streptococci (GBS) in their rectum or vagina. Half of their newborns acquire GBS at birth; 98 percent are said to be asymptomatic. Phospholipids from the GBS cell wall cause pulmonary hypertension in experimental animals. When exposed to penicillin, Streptococcus mutans immediately releases phospholipids. We hypothesize that newborns colonized with GBS receive bacterial phospholipids leading to pulmonary hypertension and respiratory distress, especially newborns of penicillin-treated mothers. Analysis of data from 1,610 colonized newborns of more than 32 weeks' gestation without early-onset disease showed that 8.8 percent had signs of respiratory distress within 48 hours after birth—an effect enhanced by penicillin use during labor. Our findings support the association of neonatal respiratory distress with asymptomatic GBS colonization and penicillin use during labor. A prospective study to relate the levels of serum bacterial phospholipids to the occurrence of respiratory distress in newborns of mothers colonized by GBS, and the effects of penicillin use during labor on the occurrence of respiratory distress, is under way. We are developing an assay to measure bacterial phospholipids in biological specimens in mothers and newborns.

Lineage ST-17 complex group B streptococci are more virulent than other serotype III

Lin

Phylogenetic lineage studies have demonstrated that serotype III GBS associated with human disease derive largely from two distinct lineages. We compared the phylogenetic lineages of invasive type III group B streptococci with those of colonizing strains in order to determine lineages associated with invasive disease. Isolates from 29 infants with early-onset disease and from 196 colonized infants, collected in a prospective, multicenter study, were assigned a sequence type (ST) by multilocus sequence typing. Overall, 54.5 percent of the isolates were in the ST-19 complex and 40.4 percent in the ST-17 complex. Invasive strains were more likely to be in the ST-17 complex than were colonizing strains (59 percent versus 38 percent). After adjusting for potential confounders, we discovered that the ST-17-complex was more likely to be associated with early-onset disease than were other lineages. These data support the hypothesis that ST-17 complex GBS are more virulent than other serotype III GBS.

Lin FY, Whiting A, Adderson E, Takahashi S, Dunn DM, Weiss R, Azimi PH, Philips JB, Weisman LE, Regan J, Clark P, Rhoads GG, Frasch CE, Troendle J, Moyer P, Bohnsack JF. 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.

Heparin-mimetic sulfated oligoxylan inhibitors of malaria parasites

Stone, Chen; in collaboration with Milhous, Sacci

When administered in children, heparin is known to inhibit the rosetting and cytoadhesion of parasitized Plasmodium falciparum erythrocytes (PfRBC) to normal red blood cells (RBCs) and the endothelium and to clear blockage of the microcirculation and ameliorate life-threatening symptoms of cerebral malaria; it also inhibits the initial malaria parasite invasion of hepatocytes (and possibly RBCs). Malaria ranks in the top three deadliest diseases globally (approximately 300 million clinical cases per year). With 1 to 3 percent of Pf parasites highly virulent, malaria causes severe and cerebral malaria and the death of about 2 million people per year (90 percent young children). There is no preventive vaccine, and malarial parasites are increasingly resistant to anti-malarial drugs. Using our library of sulfated oligoxylans, we previously applied a macrocombinatorial strategy to study heparin inhibition in vitro. We prepared the sulfated oligoxylans from a heparin/heparan sulfate (H/HS)-mimetic pharmaceutical that mimics numerous discrete biological actions of the heparin family (see below) and tested the differential potencies of sulfated oligoxylans (S-OligoS) to inhibit the capacity of P. vogelii sporozoites (freshly isolated from infected mosquitoes) to invade hepatocytes; the highest potency and concentration dependence resided in two S-OligoS in a mass class of about 7,200 and 3,700 with 43 and 56 percent inhibition at 3.5 and 5 μM, respectively. The structure of the inhibitor sequence is not clear. Current structural considerations would accommodate the presence of a maximum of six and three putative D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motifs with O-methyl groups on up to 35 percent of the GlcA moieties, respectively. We plan to test for the presence and importance of the putative motif by using capillary HPLC-mass spectroscopy of S-oligoS. Studies on the inhibition of the erythrocyte invasion stage of malarial parasites revealed that S-OligoS of mass class less than 4,500 exhibited very low inhibitory capacity while high potency was associated with S-OligoS of relatively high mass class equal to or greater than 10,000. Our data suggest differences in the molecular reactions underlying the inhibition of the two parasite stages. We are continuing to enlarge of our H/HS-mimetic S-OligoS library to generate specific anti-malaria components.

Recent advances in malaria research include the first method for generating sporozoites (and other stages of the malaria parasite) in a cell-free culture. This method now promises to provide larger amounts of sporozoites in a time short enough to allow completion of full dose-response measurements of groups of S-oligoS in bioassays by comparison. Although our project has seen limited activity this year because of resource constraints, we plan to resume our studies on inhibition of sporozoites, parasitized RBCs, and rosetting as a path toward stable, inexpensive heparin-mimetic anti-malarials against initial infection, pathologies, and/or acute cerebral malaria. We also plan experiments to elucidate putative protein ligands by using a modified gel-shift analysis of heparin oligoS-protein binding and/or to identify protein ligands by using fluorescent receptors (library of S-oligoS and/or heparin-oligoS).

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

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

A highly active heparin-mimetic inhibitor of HIV-1 entry in vitro and free of anti-thrombin toxicity (PK II, SOLIS) is an outcome of preclinical studies generated in the 1990s by our basic research findings and knowledge of the molecular and chemical glycobiology of the heparin/heparan sulfate class of polysaccharides (H/HS) and how that class's structural diversity provides the ability to modulate functions of diverse proteins in normal and disease processes (e.g., cell growth, secretion, development, blood coagulation, and infections by virus and other human pathogens). Given the diversity of sequences within H/HS chains, libraries of unique H/HS oligoS have not been readily obtainable for research. We isolated Pk II from a heparin-mimetic pharmaceutical comprising a mixture of S-oligoS. We devised scaled-up procedures to enable the clinical preparation of PK II (SOLIS) and developed a macrocombinational-type strategy to obtain a library of heparin-mimetic S-oligoS for researchers (see above). The latter enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium formation by PK II were governed by a degree of discrete structural specificities, as was the lack of anti-thrombin coagulation-inhibitory activity, two essential indicators of usefulness for further drug development.

Solis, a potential HIV-1 binding/fusion inhibitor. The number of HIV-infected Americans is approaching 1 million; AIDS morbidity and mortality worldwide (estimated at 50 million cases and 16 million deaths since the 1980s) are increasing. Despite nearly two decades of global efforts, neither a cure nor fully effective vaccine is imminent. Constant daily treatments of AIDS patients with several anti-retroviral drugs has been successful in developed countries for long-term control of viral load and deadly effects, but with a high cost in toxicity, sensitivity, and mutation to multidrug resistant strains (found in adults and children), which increasingly limit the armamentarium's efficacy. The FDA has approved 26 drugs, 16 against genomic replication (reverse transcriptase), nine against protease, and one against entry (a peptide that prevents viral fusion to the CD4 cell membrane by blocking changes in conformations of the fusion subunit of the viral envelope protein gp41). Broad consensus counsels that inhibitors against viral components that play other roles in HIV-1 attack are critically needed. We are completing a clinical preparation of SOLIS for a small Phase I safety trial. SOLIS would be an adjunct drug against binding and entry of virus into target cells (e.g., it inhibits CD4 cell adherence to gp120-coated tissue culture plates and prevents syncytium-forming fusion of HIV-1 [gp41] with CD4 cell membrane). Heparin has long been known to modulate conformations of proteins and peptides. Our heparin studies suggest that inhibition of the conformational changes in gp41 that are required for fusion progression is a possible mechanism of action in SOLIS and, if so, would be relatively insensitive to virus mutation. The laboratory is in a unique position to test such a hypothesis given our library of S-oligoS. Pk II, however, also inhibits cytopathic effects of HIV-1, suggesting that such S-oligoS might interfere with other critical elements of the virus's attack in addition to binding/entry.

¹ Oak Ridge Senior Fellow Program at NIH

COLLABORATORS

McDonald K. Horne, MD, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Maria O. Longas, PhD, Purdue University, Hammond, IN
James McMahon, PhD, Molecular Targets Development Program, NCI, Frederick, MD
Wilbur K. Milhous, PhD, Walter Reed Army Institute of Research, Washington, DC
Mark A. Miller, MD, Fogarty International Center, NIH, Bethesda, MD
John Sacci, PhD, University of Maryland, Baltimore, MD
Joseph Shiloach, PhD, Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD
Alfred L. Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD

For further information, contact rssobbinsj@nichd.nih.gov.