Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs), serve as both essential virulence factors and protective antigens. The age-related and T cell–independent immunogenicity of CPSs limit their use as vaccines, especially in infants and young children. LPSs are too toxic to be administered. Accordingly, the O-specific polysaccharides (O-SPs) of LPSs, which share the virulence-promoting and protectiveness properties of CPSs, must be purified. However, O-SPs are too small to be immunogenic (haptens). Covalent binding of CPSs or O-SPs to medically useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence on these saccharides. Part of our work focuses on making synthetic vaccines that improve immunogenicity.
Human response to polysaccharide/oligosaccharide/peptide-protein conjugate and toxoid vaccines
Schneerson, Robbins, Ben-Menachem, Chu, Coxon, Guo, Jin, Kubler-Kielb, Liu, Majadly, Mocca, Pozsgay, Trinh, Levi; in collaboration with Ashkenazi, Bellanti, Bohach, Casadevall, Claesson, Freedberg, Glatman-Freedman, Gottfredsson, Hoogerhout, Krause, Lagergard, Leppla, Marques, Miller L, Miller M, Nguyen, Nussenzweig, Passwell, Shiloach, Singer, Trollfors, Vinogradov
Shigellae. We found that the O-SP of Shigella sonnei bound to recombinant nontoxic P. aeruginosa exoprotein A (rEPA) and of S. flexneri 2a bound to the succinylated exoprotein A (rEPA-succ) was safe and induced IgG antibodies to the homologous LPS in one- to four-year-olds. A randomized, blinded Phase III study of the conjugates in this age group with each conjugate serving as a control for the other is in progress.
CROSS-REACTING POLYSACCHARIDES OF HAEMOPHILUS INFLUENZAE TYPE B (HIB) AND B. PUMILUS. A study of 10-year-old children injected in infancy with Hib-TT showed a serum level of about 4 mcg antibody/ml of IgG anti–Hib with no change in level after a DT booster but with a four-fold increase in IgG anti–TT. To investigate if concurrent administration of a cross-reacting polysaccharide with a homologous CPS offers an advantage over the use of the homologous CPS alone, we isolated the cell-wall polysaccharide (PS) of Bacillus pumilus Sh18, which cross-reacts with Hib CPS. Using gas chromatography–mass spectrometry, NMR, fast-atom bombardment, and several sugar-degrading techniques, we investigated the PS structure and found that it was composed of polyribitolphosphate substituted at C2 with N-acetyl glucosamine and polyglycerolphosphate. In addition to its anti–Hib reactivity, the PS cross-reacted with anti–Staphylococcus epidermidis known to contain polyribitol phosphate (poly Rib-P) in its teichoic acid. We conjugated the Sh18 polysaccharide to carrier proteins and evaluated its immunogenicity in mice. Conjugate-induced antibodies reacted with the homologous and several cross-reacting polysaccharides. We also synthesized poly (Rib-P) to investigate its cross-reactivity with poly(Rib-P)-containing polysaccharides.
NEISSERIA MENINGITIDIS GROUP A. N. meningitidis group A (Men. A) causes endemic and epidemic meningitis, notably in the meningitis belt of Africa. An effective and available CPS vaccine is underused. To improve its immunogenicity, we conjugated Men. A CPS to bovine serum albumin (BSA) by using ADH as a linker. In contrast to CPS alone, the conjugates proved immunogenic in mice, with booster responses after the second and third injections and bactericidal activity related to IgG anti–CPS levels. Conjugates of the cross-reactive polysaccharides E. coli K93 and B. pumilus Sh17 did not induce anti–Men. A CPS antibodies. Outbreaks of meningitis due to N. meningitides group W135 were reported recently in West Africa, Saudi Arabia, Europe, and Australia. Similar to other polysaccharides, W135 CPS is not immunogenic in young children. We prepared conjugates of this CPS bound to the recombinant Staphylococcus aureus enterotoxin C1 (rSEC), with ADH as a linker. The conjugates induced IgG anti–CPS in mice, with booster responses upon reinjection, whereas the CPS alone did not. The conjugates also induced neutralizing SEC antibodies. Injection of combined Group A and W135 conjugates induced antibody levels comparable to those of each conjugate alone.
GROUP B NEISSERIA MENINGITIDIS AND ESCHERICHIA COLIK1. Meningitis caused by Group B N. meningitidis (GBM) or Escherichia coli K1 continues to cause serious diseases that are difficult to treat; moreover, there is no licensed vaccine for their prevention. The polysaccharide alpha (2-8) N-acetylneuraminic acid (PSA) is the capsular polysaccharide of GBM and of E. coli K1 and a component of mammalian glycopeptides. Thus, it is a “self-antigen,” and there is concern that vaccines designed to induce PSA immune responses may cause immunopathology. Although antibodies to PSA bind to human tissues in vitro, they have not been shown to induce pathology in vivo. The incidence, severity, and nature of systemic infection caused by GBM are similar to those caused by the closely related group C meningococci (GCM). As shown for other polysaccharides, age-related acquisition of serum PSA antibodies occurs in humans. Purified PSA is not immunogenic; however, as a component of bacteria or when bound to proteins as a conjugate, this CPS induces antibodies of the three major isotypes, with its IgM and IgG components protective both in vitro and in vivo. Despite the CPS’s similarity to mammalian glycoproteins, no data exist to indicate an association of IgG anti–PSA with immunopathology in experimental animals or in humans. In collaboration with Mark Miller and members of the Lanspitali University Hospital and Statens Serum Institut, we are conducting a long-term examination of survivors of groups B and C meningococcal meningitis to uncover any unusual diseases that are considered autoimmune. We propose consideration of clinical trials of PSA conjugate vaccines shown to be immunogenic and protective in animals.
BORRELIA BURGDORFERI. B. burgdorferi, a spirochete transmitted through the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against the spirochete is not effective under 12 years of age and was recently taken off the market. LPS has been described in other spirochetes, but its presence in B. burgdorferi is still debated; we have not been able to confirm its presence. However, our search for LPS revealed two unique glycolipids: cholesteryl 6-O-acyl-beta-D-galactopyranoside (BbGL I) and 1,2-di-O-acyl-3-O-alpha-D-galactopyranosyl-sn-glycerol (BbGL II). We found evidence that the two glycolipids are surface-exposed. Injected in various formulations into mice, BbGL I induced specific antibodies. Our investigation of the effect of BbGL-I and BbGL-II on human PMNs and monocytes showed that the glycolipids induced secretion of increased levels of IFN-gamma, IL-4, and TNF-alpha.
BACILLUS ANTHRACIS. B. anthracis, a cause of lethal human infection and a potential bioterrorist weapon, exhibits two essential virulence factors, without either of which it is not pathogenic for humans. The factors are the anthrax toxin and the capsule. The toxin is composed of three proteins: Lethal Factor (LF), Edema Factor (EF), and Protective Antigen (PA), each by itself nontoxic. PA binds to mammalian cells, and a 20 kDa peptide must be hydrolyzed off PA, exposing a site to which LF or EF may bind and rendering toxins that enzymatically modify substrates in the mammalian cell cytosol. The capsule, composed of poly-D-gamma-glutamic acid (PGA), is non-immunogenic and of unknown protective effect. Although the licensed vaccine is safe and protective, its limitations justify development of improved vaccines. Thus, we isolated a recombinant PA from an uncapsulated strain. We found that several formulations with formaldehyde-treated and alum-adsorbed materials were immunogenic in mice; therefore, we have begun clinical evaluation of the formulations. Using another approach, we isolated the capsule from a non-toxic strain and bound it or corresponding synthetic peptides to BSA, rEPA, or rPA. To identify the optimal lengths of construct peptides, we conjugated 5-, 10-, and 20-mers of D or L configuration with active groups at the C- or N-terminus. We characterized the conjugates by physicochemical and immunological assays, the latter in five- to six-week-old mice. In contrast to PGA alone, all conjugates were immunogenic, and the antibodies induced by D-PGA were opsonophagocytic. We found that rPA was the most effective carrier. Conjugates produced by additional conjugation methods yielded conjugates equally immunogenic in mice.
SEROSURVEY OF ARMED FORCES PERSONNEL IMMUNIZED WITH THE LICENSED ANTHRAX VACCINE. We made a survey of IgG antibodies in 246 sets of paired sera taken from Armed Forces personnel immunized with the FDA-licensed Anthrax Vaccine Adsorbed (AVA) and stored in the Serum Bank of the U.S. Armed Forces Repository. Using ELISA and purified PA from Bacillus anthracis, we assayed paired sera from HIV-negative personnel after the third, fourth, and sixth injections. The personnel were stratified according to sex and age (18 to 24 years and over 24 years). We considered as pre-immune sera from before the third injection. We found serum conversion rates equal to or greater than four-fold in antibody levels as follows: 85 percent after the third injection, 68 percent after the fourth, and 45 percent after the sixth. The levels in males were similar to those in females but statistically significantly higher in the younger compared with the older group. We will use these values to evaluate the IgG anti–PA levels induced by our investigational B. anthracis vaccines.
PLASMODIUM FALCIPARUM. Malaria is a leading cause of morbidity and mortality worldwide, especially in children, and is estimated to cause over 1 million childhood deaths annually. P. falciparum causes the most severe form of disease. Experimental vaccines have been described and some tested clinically, but no licensed vaccine is available. The most studied vaccine contains the circumsporozoite protein (CS), which is expressed extracellularly on the sporozoite, and various forms of NANP, its synthesized repeat. The vaccines are safe and immunogenic but poorly protective, even when administered with adjuvants. Based on our studies with peptides of the B. anthracis capsule, we synthesized peptides of four-repeat units of NANP and bound them to carrier proteins, studying their immunogenicity in mice without adjuvants and by a scheme suitable for humans. Preliminary results showed high levels of antibodies, with circumsporozoite-neutralizing activity roughly correlated with levels measured by ELISA. In another approach to providing a transmission-blocking vaccine, we bound the low-molecular–weight protein PFS25 to carrier proteins and injected it into mice to evaluate their antibody responses.
BORDETELLAE. B. parapertussis causes pertussis in humans, though in a milder form and with lower frequency than B. pertussis. B. bronchiseptica causes respiratory infection in animals but rarely in humans. B. parapertussis and B. bronchiseptica do not produce pertussis toxin, but their LPS structures are reportedly similar. We purified the LPS of B. parapertussis and of two strains of B. bronchiseptica and isolated their O-SPs, whose structural and immunological properties are now under study.
HAEMOPHILUS DUCREYI. H. ducreyi is the cause of chancroid, a sexually transmitted disease characterized by painful genital ulceration. Importantly, chancroid enhances the spread of HIV infection. A major virulence factor and a potentially protective antigen of H. ducreyi is its lipo-oligosaccharide (LOS). We isolated the LOS, hydrolyzed its lipid A, and bound the O-SP to carrier proteins by two new methods. As for the first method, we bound the carbonyl group at the KDO of the reducing end of the O-SP to an amino-oxy group of a bifunctional linker (prepared in our laboratory) and bound the linker to carrier proteins. As for the second method, we used adipic acid dihydrazide to bind to both the carbonyl group of the O-SP and benzaldehyde-derivatized carrier proteins. The conjugates preserved the external, nonreducing end of the O-SP and precipitated with H. ducreyi antisera. Their immunogenicity in mice is under investigation.
REPLACEMENT OF THE DIPHTHERIA AND PERTUSSIS COMPONENTS OF DTP WITH GENETICALLY INACTIVATED MUTANT TOXINS. Acellular pertussis vaccines have considerably reduced the systemic reactions formerly observed with cellular vaccines of DTP but have not eliminated the extensive swelling (sometimes involving an entire limb) observed after the fifth injection of DTaP. This reaction, an Arthus hypersensitivity resulting from high levels of antibodies reacting with DTaP, could discourage the vaccine’s use in adults who serve as the major reservoir of pertussis for infants. A critical level of pertussis toxin (PT) antibodies is both essential and sufficient to prevent infection with B. pertussis. We have pointed out the similarities between the immunity induced by diphtheria and pertussis toxoids. Genetically inactivated pertussis and diphtheria toxins are more immunogenic and therefore induce comparable levels of anti-toxin at lower protein levels than the formalin-treated native toxins. In addition, the chemically inactivated pertussis toxoids exert mild but constant immunosuppressive activity. Replacement of the D and aP components with the improved antigens will reduce the amount of protein in DTaP and, most likely, the incidence and severity of local reactions in teenagers and adults.
Claesson BA, Trollfors B, Lagergard T, Knutsson N, Schneerson R, Robbins JB. Antibodies against Haemophilus influenzae type b capsular polysaccharide and tetanus toxoid before and after a booster dose of the carrier protein nine years after primary vaccination with a protein conjugate vaccine. Pediatr Infect Dis J 2005;24:463-464.
Glatman-Freedman A, Casadevall A, Dai Z, Jacobs WR, Li A, Morris SL, Navoa JA, Piperdi S, Robbins JB, Schneerson R, Schwebach JR, Shapiro M. Antigenic evidence of prevalence and diversity of Mycobacterium tuberculosis arabinomannan. J Clin Microbiol 2004;42:3225-3231.
Jin Z, Bohach GA, Shiloach J, Norris SE, Freedberg DI, Deobald C, Coxon B, Bobbins JB, Schneerson R. Conjugates of groups A and 135 capsular polysaccharides of Neisseria meningitis bound to recombinant Staphylococcus aureus Enterotoxin C1: preparation, physico-chemical characterization, and immunological properties in mice. Infect Immun 2005;73:7887-7893.
Robbins JB, Schneerson R. Future vaccine development at NICHD. Ann NY Acad Sci 2004;1038:49-59. Robbins JB, Schneerson R, Gotschlich EC. Surveillance for bacterial meningitis by means of polymerase chain reaction. Clin Infect Dis 2005;40:26-27.
Robbins JB, Schneerson R, Trollfors B, Sato H, Sato Y, Rappuoli R, Keith JM. The diphtheria and pertussis components of diphtheria-tetanus toxoids-pertussis vaccine should be genetically inactivated mutant toxins. J Infect Dis 2005;191:81-88.
Stein DM, Robbins J, Miller MA, Lin FY, Schneerson R. Are antibodies to the capsular polysaccharide of Neisseria meningitidis group B and Escherichia coli K1 associated with immunopathology? Vaccine 2005 [Epub ahead of print].
Other conjugate-induced polysaccharide vaccines
Szu, Schneerson, Li, Ftacek, Korzeniowska-Kowal, Hunt; in collaboration with Ahmed, Bohach C, Clements, Hanson, Lin, Moore, Parke, Prentice, Shiloach
We enhance the immunogenicity of capsules or lipopolysaccharides by binding them to carrier proteins.
SALMONELLA TYPHI. Sequential clinical studies in adults and children in Vietnam, an area with a high attack rate of typhoid, showed that vaccines of the capsular polysaccharide (Vi) of S. typhi conjugated to the recombinant Pseudomonas aeruginosa exoprotein A (rEPA) were safe and elicited high serum IgG antibody levels to the Vi. A Phase III trial of the conjugate vaccine in about 12,000 two- to five-year-olds showed an efficacy of Vi-rEPA of 89 percent after 48 months of surveillance. We estimated the minimal protective level of serum IgG anti-Vi to be 3.7 ELISA units. We determined optimal dosage in a study conducted in Northern Vietnam in two- to five-year-olds. A clinical trial of Vi-rEPA injected with DTP in Vietnam has been approved and will shortly commence.
SALMONELLA PARATYPHI A. S. paratyphi A is the second most common cause of enteric fever in Southeast Asia. We found that our conjugate vaccine of the O-SP polysaccharide of S. paratyphi A and rEPA was safe and immunogenic in adults, teenagers, and two- to four-year-olds. We also found that the O-acetyl content of the PS was directly related to the immunogenicity of the conjugate. In Southeast China, the number of cases of S. paratyphi A has exceeded thee number of cases of S. typhi. A Phase III clinical trial is planned in China with S. paratyphi A O-SP-rEPA conjugate vaccine manufactured by the Chinese under NIH guidance.
ESCHERICHIA COLI O157.E. coli O157 is an emerging pathogen causing hemorrhagic colitis and hemolytic uremic syndrome, especially in young children. A Phase I study of E. coli O157 O-specific polysaccharide-rEPA conjugate demonstrated safety and immunogenicity in adult volunteers. In a Phase II study of the conjugate, 55 two- to five-year-olds were injected once or twice. We found that the vaccine was safe and immunogenetic; 98 percent of children responded with a greater than four-fold rise of IgG anti–LPS six months after the first injection but exhibited no booster response following a second dose six weeks after the first. We purified genetically detoxified Shiga toxins I and II from a mutant E. coli O157 and will conjugate them to the O-SP to make a bivalent vaccine. Given that the major reservoir of E. coli O157 is cattle, we tested an LPS-protein conjugate vaccine in cattle and found that it was safe and immunogenic. A challenge study showed an inverse correlation between the serum IgG anti-LPS level and the degree of colonization with E. coli O157.
ENTEROTOXIGENIC ESCHERICHIA COLI. Only a few of the many O-serotypes of E. coli, including enterotoxigenic E. coli (ETEC), cause an illness commonly called Traveler’s Diarrhea in most countries. The illness’s incidence and associated morbidity and mortality are, however, highest in developing countries. ETEC can secrete either an exotoxin similar in structure and action to cholera toxin and known as the heat-labile toxin (LT) or a polypeptide of 19 amino acids called the stable toxin (ST). About 40 percent of ETEC in strains from patients secrete LT alone, 20 percent secrete both LT and ST, and 60 percent secrete ST alone. Despite the availability of good information from animal models, scientists have not studied either the immunogenicity or protective actions of LT administered parenterally and instead have drawn conclusions about the protective activity of LT antibodies from animals injected with LT admixed with Freund’s adjuvants, from humans infected with ETEC, or from clinical trials with cross-reactive cholera toxin. The limitations of these approaches to evaluating the usefulness of LT as a vaccine are well known. To investigate further LT’s potential protective immunity, we obtained purified LT and a recombinant mutant non-toxic protein (rLT) from John Clements. After treating both with low concentrations of formaldehyde, as is done for diphtheria and pertussis toxin cross-reacting mutants, we injected the wild-type or rLT into young outbred mice and evaluated their immunogenicity. The treated rLT was the most immunogenic material. We are now studying the effect of adsorbing these proteins onto aluminum hydroxide. We plan to modify the disulfide bonds of a synthetic ST to reduce toxicity and improve solubility. Studies are under way to develop conjugates of ST with LT or rLT to improve immunogenicity. Our overall objective is to evaluate the clinical effect of parenteral administration of LT and a conjugate of LT with ST for prevention of Traveler’s Diarrhea.
VIBRIO CHOLERA. V. cholera O1 and O139 are the major serotypes found in cholera infections. Conjugates synthesized with the capsular polysaccharide of O139 and the O-SP of LPS of O1 elicited vibriocidal antibodies in mice. We have used detoxified LPS from both O1 Inaba and Ogawa serotypes. In mice, the conjugates elicited higher antibody levels than did LPS alone. We plan to make synthetic O-SP of Ogawa, conjugate it to a protein carrier, and compare the immunogenicity of cholera vaccines prepared with native or chemically synthesized O-SP in animal studies. V. cholera O139, similar to O1, secrets cholera toxin and causes sporadic cholera outbreaks in Southeast Asia. Unlike serotype O1, type O139 contains a capsule. We found that convalescent human sera contained high levels of vibriocidal IgM antibodies against the capsule. IgM secretion is usually short-lived, and the immunoglobulin does not penetrate the gut lumen to lyse the organisms. We conjugated V. cholera O139 CPS to H21G, a mutant diphtheria toxin. In mice, this conjugate showed high levels of vibriocidal IgG antibodies. We plan a phase I study of monovalent conjugate vaccines and a trivalent vaccine against these major V. cholera strains.
SALMONELLA TYPHIMURIUM. S. typhimurium causes most salmonella infections in developed countries. Antibiotic-resistant strains are common among disease isolates. We compared the O-specific polysaccharide structures of two of the most common and antibiotic-resistant strains. We found that the O-acetyl group on the abequose was a major antigenic site and an immunodominant determinant. Strains that are O-acetyl–positive reacted poorly with antibodies from O-acetyl–negative strains. Selection of strains for a vaccine will therefore rely on an epidemiologic survey and bacteriologic identification.
CAMPYLOBACTER JEJUNI. C. jejuni infection is one of the most common enteric infections. Campylobacter is microaerophilic, and fermentation in liquid media has been difficult. We studied various media and fermentation conditions and established requirements for growth in liquid media. SDS-PAGE silver staining of the bacterium’s purified LPS indicated that it is a lipo-oligosaccharide (LOS). Chemical analysis of the O-SP showed keto groups and no sialic acid. Conjugate vaccines prepared with LOS or with de-acylated LOS were immunogenic, and antibodies elicited by the vaccines showed bactericidal activity in the presence of complement.
THE EFFECT OF LOW BIRTH WEIGHT ON THE IMMUNE RESPONSE IN LATE ADULTHOOD. Substantial evidence links small size at birth (not due to prematurity) to susceptibility to chronic disease in later life. Some components of the immune function may be programmed in early life. We investigated the association between birth size and response to vaccination with Vi-rEPA in a cohort of 257 adults (mean age: 29.4 years; 146 men) born in an urban slum in Lahore, Pakistan, between 1964 and 1978; we are currently evaluating the results.
Canh DG, Lin FY, Thiem VD, Trach DD, Trong ND, Mao ND, Hunt SW, Schneerson R, Robbins JB, Chu C, Shiloach J, Bryla DA, Bonnet M, Schulz D, Szu SC. Effect of dosage on the immunogenicity of Vi conjugate vaccine injected twice in 2- to 5-year-old Vietnamese children. Infect Immun 2004;72:6586-6588.
Moore SE, Jalil F, Ashraf R, Szu SC, Prentice A, Hanson I. Birth weight predicts response to vaccination in adults born in an urban slum in Lahore, Pakistan. Am J Clin Nutr 2004;80:453-459.
COLLABORATORS
Amina Ahmed, MD, Carolina Medical Center, Charlotte, NC
Shai Ashkenazi, MD, Schneider Children’s Hospital, Petah Tikva, Israel
Joseph Bellanti, MD, Georgetown University Medical Center, Washington, DC
Carolyn H. Bohach, PhD, University of Idaho, Moscow, ID
Gregory Bohach, PhD, University of Idaho, Moscow, ID
Arturo Casadevall, MD, PhD, Albert Einstein College of Medicine, New York, NY
Bo Claesson, MD, Sahlgrenska Universitetssjukuset, Göteborg, Sweden
John Clements, PhD, Tulane University Health Sciences Center, New Orleans, LA
Daron I. Freedberg, PhD, Laboratory of Biophysics, CBER, FDA, Bethesda, MD
Aharona Glatman-Freedman, MD, Albert Einstein College of Medicine, New York, NY
Magnus Gottfredsson, MD, Landspítali Haskolasjukrahus, Reykjavik, Iceland
Lars Hanson, MD, Göteborgs Universitet, Göteborg, Sweden
Peter Hoogerhout, PhD, Nederlands Vaccin Instituut, Bilthoven, Netherlands
Tyra Grove Krause, PhD, Statens Serum Institut, Copenhagen, Denmark
Teresa Lagergard, PhD, Göteborgs Universitet, Göteborg, Sweden
Stephen H. Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
Kimi F. Lin, MD, Epidemiology Branch, NICHD, Bethesda, MD
Adriana Marques, MD, Laboratory of Clinical Infectious Diseases, NIAID, Bethesda, MD
Louis Miller, MD, Malaria Vaccine Development Branch, NIAID, Bethesda, MD
Mark Miller, MD, Fogarty International Center, NIH, Bethesda, MD
Sophie E. Moore, PhD, London School of Hygiene and Tropical Medicine, London, UK
Nga Y. Nguyen, PhD, Core Facility, CBER, FDA, Bethesda, MD
Ruth Nussenzweig, MD, PhD, New York University Medical Center, New York, NY
James C. Parke, Jr., MD, Carolina Medical Center, Charlotte, NC
Justen H. Passwell, MD, Chaim Sheba Medical Center, Tel-Hashomer, Israel
Andrew M. Prentice, MD, London School of Hygiene and Tropical Medicine, London, UK
Joseph Shiloach, PhD, Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD
Darrell Singer, MPH, Walter Reed Army Institute of Research, Washington, DC
Birger Trollfors, MD, Sahlgrenska Universitetssjukuset, Göteborg, Sweden
Evgeny Vinogradov, PhD, Institute for Biological Sciences, National Research Council, Ottawa,Canada
For further information, contact yxr@cu.nih.gov.