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Establishing a correlate of protection is essential for the development and licensure of Shigella vaccines. We examined potential threshold levels of serum IgG to Shigella lipopolysaccharide (LPS) that could predict protection against shigellosis.We performed new analyses of serologic and vaccine efficacy (VE) data from two randomized vaccine-controlled trials of the Shigella sonnei-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate conducted in young adults and children aged 1-4 years in Israel. Adults received either S. sonnei-rEPA (n = 183) or control vaccines (n = 277). Children received the S. sonnei-rEPA conjugate (n = 1384) or S. flexneri 2a-rEPA conjugate (n = 1315). VE against culture-proven shigellosis was determined. Sera were tested for IgG anti-S. sonnei LPS antibodies. We assessed the association of various levels of IgG anti-S. sonnei LPS antibodies with S. sonnei shigellosis risk using logistic regression models and the reverse cumulative distribution of IgG levels.Among adults, four vaccinees and 23 controls developed S. sonnei shigellosis; the VE was 74% (95% CI, 28-100%). A threshold of ≥1:1600 IgG anti-S. sonnei LPS titre was associated with a reduced risk of S. sonnei shigellosis and a predicted VE of 73.6% (95% CI, 65-80%). The IgG anti-S. sonnei LPS correlated with serum bactericidal titres. In children, a population-based level of 4.5 ELISA Units (EU) corresponding to 1:1072 titre, predicted VE of 63%, versus 71% observed VE in children aged 3-4 years. The predicted VE in children aged 2-4 years was 49%, consistent with the 52% observed VE.Serum IgG anti-S. sonnei LPS threshold levels can predict the degree of VE and can be used for the evaluation of new vaccine candidates.

Shigella flexneri (S. flexneri) 6 has emerged as an important cause of shigellosis. Our efficacy study of Shigella sonnei and S. flexneri 2a O-specific polysaccharide (O-SP) conjugates in 1–4 year-olds had too few S. flexneri 2a cases for efficacy evaluation but surprisingly showed protection of 3–4 year-olds, S. flexneri 2a-recipients, from S. flexneri 6 infection. To investigate this cross-protection antibodies to both Shigella types were investigated in all sera remaining from previous studies. Twenty to 30% of 3–44 year-old humans injected with S. flexneri 2a conjugate responded with ≥4-fold increases of IgG anti type 6, p < 0.00001. The specificity of these antibodies was shown by inhibition studies. S. flexneri 6 infection of 2 children induced besides S. flexneri 6, also S. flexneri 2a antibodies, at levels of S. flexneri 2a vaccinees. S. flexneri 2a antibodies induced by S. flexneri 6 conjugates could not be studied since no such conjugate was assessed in humans and mice responded almost exclusively to the O-SP of the injected conjugate, with no cross-reactive antibodies. Our results indicate induction of cross-reactive protective antibodies. The O-acetylated disaccharide shared by S. flexneri 6 and 2a O-SPs, is the likely basis for their cross-reactivity. S. flexneri 6 O-SP conjugates, alone and in combination with S. flexneri 2a, merit further investigation for broad S. flexneri protection.

Combination vaccines containing a monocomponent acellular pertussis (aP) vaccine, manufactured at Statens Serum Institut (SSI), Denmark, have successfully controlled Bordetella pertussis infections in Denmark since 1997. The efficacy of this aP vaccine was 71% in a double-blind, randomised and controlled clinical trial. Its safety and immunogenicity have been demonstrated in infants, children, adolescents and adults. In approximately 500,000 children it was effective against pertussis requiring hospitalisation (VE: 93% after 3 doses) and against pertussis not requiring hospitalisation (VE: 78% after 3 doses). IgG antibodies against pertussis toxin (IgG anti-PT) response rates after booster vaccination of adults with tetanus, diphtheria and aP combination vaccine (TdaP) were considerably higher for this monocomponent aP vaccine containing 20μg pertussis toxoid, inactivated by hydrogen peroxide (92.0%), than for two multicomponent aP vaccines inactivated by formaldehyde and/or glutaraldehyde: 3-component aP with 8μg pertussis toxoid (77.2%) and 5-component aP with 2.5μg pertussis toxoid (47.1%), without compromising the safety profile. In Denmark where this monocomponent aP vaccine has been the only pertussis vaccine in use for 15 years, there has been no pertussis epidemic since 2002 (population incidence 36 per 100,000), in contrast to neighbouring countries, where epidemics have occurred. This monocomponent aP vaccine can be used in combination vaccines for primary and booster vaccination against pertussis in all age groups and is an important tool for successful pertussis control.

Recent data showing the high incidence of typhoid fever in young children, the demonstration of safety and efficacy of a Vi conjugate for this age group, the safety and similar immunogenicity in infants when administrated concurrently with EPI vaccines, together with the interests of manufacturers and investigators in studying such conjugate vaccines prompted us to prepare a human IgG anti-Vi standard to facilitate this work. Volunteers were injected with an investigational Vi-recombinant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) conjugate vaccine. Plasmas with the highest levels of IgG anti-Vi were pooled. The IgG anti-Vi content of this preparation, assayed by precipitin analysis with purified Vi, was 33 μg/ml. Accordingly, the estimated IgG anti-Vi protective level of 3.5 ELISA unit/ml, derived from our efficacy trial of Vi-rEPA in 2-5 years old children, is equivalent to 4.3 μg/ml. This reagent is suitable for comparison of immune response of Vi conjugate vaccines or for other purposes requiring anti-Vi measurement.

Carbapenem resistant Klebsiella pneumoniae (CRKP) are isolated with increasing frequency, especially from immunocompromized patients. The capsular polysaccharide (CPS) types of CPKP were not determined. Investigation of two CRKP isolates from a 2011 outbreak at the Clinical Center, the National Institutes of Health, identified a new capsular type shared by the two isolates, similar to K. pneumonia K19 and K34 but structurally different than any published K. pneumoniae CPS repeating unit: The LPS of the two isolates was found to have no O-specific polysaccharide and the chemical structure of the core oligosaccharides agreed with the published data. If this structure type will be prevalent among CPKP isolates, our findings could facilitate rapid diagnosis and help to develop new therapeutic solutions to this antibiotic resistant pathogen. © 2013 Elsevier Inc. All rights reserved.

TYPHOID FEVER Typhoid fever, which is transmitted to susceptible hosts by food and water contaminated with S. typhi, remains a major health problem in developing countries. Since the 1990s, the emergence of antibiotic resistant strains has made the treatment of typhoid fever more difficult (14). Asymptomatic carriers constitute the reservoir of infection (15). With no near-term solution to providing clean water and environment to all populations in developing countries, vaccination is a costeffective way to control and eliminate this disease (16-18).

We reviewed the literature that is the basis for our proposal that (2→8)-α-Neu5Ac conjugates will be safe and effective vaccines for Group B meningococci (GBMs), Escherichia coli K1, and Pasteurella haemolytica A2. Although (2→8)-α-Neu5Ac is a virulence factor and a protective antigen of these three pathogens, it is also a component of normal tissues (neural cell adhesion molecule). Natural, anti–(2→8)-α-Neu5Ac present in most adults, vaccine-induced antibodies, and even high levels of spontaneously appearing monoclonal anti–(2→8)-α-Neu5Ac did not cause autoimmunity. Although it is not possible to prove a null hypothesis, there are no epidemiologic, serologic, immunologic, or clinical data to indicate that (2→8)-α-Neu5Ac antibodies will induce pathology or an autoimmune disease. No increased pathology caused by these antibodies was found, even in neonates and infants of mothers recovered from GBM meningitis. The lack of pathology mediated by anti–(2→8)-α-Neu5Ac may be explained by different presentations of (2→8)-α-Neu5Ac on bacterial and mammalian cells and by the unusual physicochemical properties of anti–(2→8)-α-Neu5Ac. Based on clinical and experimental data collected over 30 y and because (2→8)-α-Neu5Ac is an essential virulence factor and a protective antigen for GBM, E. coli K1, and P. haemolytica A2, protein conjugates of it are easy to prepare using inexpensive and plentiful ingredients, and they would be compatible with routinely administered infant vaccines, clinical studies of these conjugates should proceed.

Typhoid fever remains a serious problem in developing countries. Current vaccines are licensed for individuals who are 5 years old or older. A conjugate of the capsular polysaccharide (CP) of Salmonella enterica serovar Typhi (Vi) bound to recombinant exoprotein A of Pseudomonas aeruginosa (Vi-rEPA) enhanced Vi immunogenicity and protected 2- to 5-year-olds in Vietnam. In this study, Vi-rEPA was evaluated for use in infants. A total of 301 full-term Vietnamese infants received Expanded Program on Immunization (EPI) vaccines alone or with Vi-rEPA or Haemophilus influenzae type b-tetanus toxoid conjugate (Hib-TT) at 2, 4, and 6 months and Vi-rEPA or Hib-TT alone at 12 months. Infants were visited 6, 24, and 48 h after each injection to monitor adverse reactions. Maternal, cord, and infant sera were assayed for IgG anti-Vi and for IgG antibodies to Hib CP and the diphtheria, tetanus, and pertussis toxins at 7, 12, and 13 months. No vaccine-related serious adverse reactions occurred. In the Vi-rEPA group, the IgG anti-Vi geometric mean (GM) increased from the cord level of 0.66 to 17.4 enzyme-linked immunosorbent assay units (EU) at 7 months, declined to 4.76 EU at 12 months, and increased to 50.1 EU 1 month after the 4th dose (95% of infants had levels of ≥3.5 EU, the estimated protective level). Controls had no increase of the IgG anti-Vi GM. Infants with cord anti-Vi levels of

No vaccine is universally active against serogroup B meningococci. A theoretical concern that serogroup B capsular polysaccharide may induce autoimmunity hampers vaccine development. We studied long-term complications in 120 survivors of meningococcal disease. No evidence of increased autoimmune, neurological, or psychiatric disease was noted., Background. Given the identity between Neisseria meningitidis serogroup B (MenB) capsular polysaccharide (polysialic acid; PSA) and PSA found on neural cell adhesion molecules, it has been proposed that infection with MenB or vaccination with PSA may be associated with subsequent autoimmune or neurological disease. Methods. We conducted 2 studies. The first was a retrospective nationwide study of invasive meningococcal disease (IMD) in Iceland (with 541 subjects) during the period 1975–2004, and we cross referenced this cohort with databases with respect to subsequent diagnosis of autoimmune disorders. A follow-up study involving 120 survivors of IMD was performed. The study included 70 patients with a history of MenB and 50 patients with N. meningitidis serogroup C (MenC) infection, who served as control subjects. Participants answered standardized questionnaires (Beck’s Depression Inventory [BDI] II, Depression Anxiety Stress Scales [DASS], and Patient Health Questionnaire [PHQ]), and serum levels of immunoglobulin (Ig) G against MenB and MenC capsular polysaccharides were measured. Results. The nationwide cohort had 9166 patient-years of follow up. No evidence of increased autoimmunity was found to be associated with MenB, compared with MenC. In the follow-up study, patients were evaluated 16.6 years after the infection, representing 2022 patient-years of observation. Comparable rates of most complications were recorded, but MenC infections were associated with arthritis (P = .008) and migraine headaches (P = .01) more frequently than were MenB infections. No difference was observed with respect to scores on BDI-II, DASS, or PHQ. IgG anti-MenB and anti-MenC capsular polysaccharide levels were not related to patient complaints. Conclusions. This study does not support the hypothesis that MenB infection may predispose to autoimmunity. MenC infections are associated with a higher prevalence of arthritis and migraine headaches. No evidence of antibody-associated pathology was detected at long-term follow-up.

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the beta-configuration, while in all other RUs the GlcNAc was present in the alpha-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.

Shigellosis, an enteric disease, is on the World Health Organization's priority prevention list. In one study, the Shigella sonnei O-specific polysaccharide (O-SP)-protein conjugate showed 72% protection against disease in Israeli army recruits exposed to high rates (8–14%) of infection. The protection was related to vaccine-induced IgG anti-O-SP levels. Synthetic oligosaccharides of Shigella dysenteriae type 1, bound by their reducing ends to a carrier protein (“sun”-type configuration), induced significantly higher antibody levels than the native O-SP bound to protein by multiple-point attachments (“lattice”-type configuration). Attempts to synthesize the S. sonnei O-SP based oligosaccharides were not successful. Here, we describe the isolation, characterization, and conjugation of low-molecular-mass O-SP-core (O-SPC) fragments. The O-SPC fragments were bound by their reducing ends similar to the preparation of the synthetic S. dysenteriae type 1 conjugates. The O-SPC conjugates used oxime linkages between the terminal Kdo residues at the reducing ends of the S. sonnei saccharides and aminooxy linkers bound to BSA or a recombinant diphtheria toxin. The coupling reaction was carried out at a neutral pH and room temperature. IgG antibody levels induced in young outbred mice by the S. sonnei O-SPC conjugates were significantly higher then those elicited by the O-SP conjugates. Accordingly, we propose to evaluate clinically these conjugates.

Background Group B meningococcal (GBM) disease induces antibodies that react in vitro with neural cell adhesion molecules in fetal brain tissue. Because IgG antibodies to GBM cross the placenta, the authors investigated whether women with a previous GBM disease had an increased risk of giving birth to preterm or to stillborn infants and whether the live-born children had an increased risk of birth defects. Methods Data were obtained from 4 national registries in the period 1974-2005 to form 2 cohorts: (1) 1422 women with confirmed GBM disease, and (2) their 502 firstborn children. Results Overall, there was no increased risk of preterm or stillbirths among the first cohort. Among the children, there was no increased risk of being born small for the gestational age, having birth defects (OR: 1.00; 95% CI: 0.53-1.90), diseases of the nervous system (HR: 0.38; 95% CI: 0.08-1.74), or any diseases within the first 3 years of life (HR: 1.06; 95% CI: 0.78-1.45) compared to births from a reference population with prior group C meningococcal disease. Conclusions The results do not support the proposal that GBM is associated with immunoreactive disease that may affect the health of the offspring and are consistent with previous findings that GBM disease is not associated with an increased risk of autoimmune disease.

Synthetic oligosaccharides composed of several repeats of Shigella dysenteriae type 1 O-specific polysaccharide (O-SP), bound by their reducing ends to a carrier protein (“sun” type configuration), induced in mice significantly higher antibody levels than conjugates of the native O-SP (“lattice” type configuration). Here we present isolation and characterization of low molecular mass oligosaccharides of Shigella sonnei lipopolysaccharides and their conjugation to several carrier proteins. Conjugates were formed by oxime linkages between the terminal Kdo residues of the reducing ends of the saccharides and aminooxy linkers bound to the protein. IgG antibody levels induced in young outbread mice by these conjugates were significantly higher than those prepared with the full length native O-SP. We propose clinical evaluation of the new S. sonnei conjugates.

The seroconversion rates and geometric mean concentrations (GMC) of IgG anti-PA for stored sera from U.S. military personnel immunized 3, 4, and 6 times with the U.S. licensed anthrax vaccine adsorbed were studied. Anti-PA IgG concentrations were measured by ELISA. All 246 vaccinees had low but detectable pre-immunization anti-PA IgG (GMC 1.83 microg/mL). Three doses elicited a GMC of 59.92 microg/mL and a seroconversion rate of 85.3%, four doses elicited a GMC of 157.44 microg/mL and 67.9% and the sixth of 276.95 microg/mL and 45.5%, respectively. The forth dose elicited 100% seroconversion compared to the pre-immunization level. These results should facilitate comparison between different immunization schedules and new vaccines.

Endemic and epidemic shigellosis, an acute invasive disease of the lower intestines, afflicts millions of people worldwide with an estimated one million fatalities per annum at a low infectious dose. Our approach to vaccine development against Shigella is based on the hypothesis that serum IgG antibodies to the O-specific polysaccharide (O-SP) domains of the LPS of these organisms confer protection to infection. The synthetic oligosaccharides corresponding to the tetrasaccharide repeating unit of the O-SP of Shigella dysenteriae type 1 covalently linked to human serum albumin elicited O-SP-specific IgG in mice. The antibody levels were a function of both the saccharide chain length and their loading on the protein. These synthetic saccharide conjugates elicited significantly higher levels of IgG anti O-SP than conjugates prepared with the O-SP from the bacteria. Here, we evaluated the influence of the nonreducing terminal monosaccharide on the serum antibody response. To this end, we prepared synthetic oligosaccharides comprising hexa- to tridecasaccharide fragments of the native O-SP, having one of the four monosaccharide residues that constitute the repeating unit at their termini and bound them to BSA by a single-point attachment. The conjugates contained an average of 19 saccharide chains per BSA. The synthetic oligosaccharides inhibited the binding of serum raised against whole bacteria to its LPS to a similar extent but lower than the native O-SP. The highest anti-LPS levels were elicited by conjugates having N -acetylglucosamine (10-mer) or galactose residues (7- and 11-mers) at their nonreducing termini.

The number of pertussis cases reported to the CDC increased from 5158 in 1995 to 21,503 in 2005. Most of the increase was in individuals greater than 10 years of age. This increase occurred also in other developed nations despite high coverage of infants and young children with the acellular pertussis vaccine. In Goteborg Sweden, virtual elimination of pertussis occurred following immunization of 70% of the children less than 10 years old with monocomponent pertussis toxoid (PTx). Immunity following disease or vaccination with either the cellular or acellular pertussis vaccine wanes gradually so that older children and adults may again become susceptible. Currently, PTx is made from chemically-inactivated pertussis toxin (PT). The most immunogenic PTx is made from genetically-inactivated mutant PT that induces higher levels of IgG anti-PT at all ages. Because of its greater immunogenicity, the genetically-inactivated PTx can be expected to be more protective on an individual and on a community basis for a longer duration than the current product. Manufacturers have declined to produce the genetically-inactivated PTx because of the expense required to change to the improved vaccine and not because of scientific issues.

Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of the lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here, we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S. flexneri type 2a LPS. [structure: see text]

The immunogenicity of Bacillus anthracis capsule (poly-γ- d -glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10 3 were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10 3 . An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10 4 following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis . Most important, the PGA-TT–induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines.

The bacterial cell-wall-associated teichoic acids contain predominantly D-ribitol residues interconnected by phosphodiester linkages. Because of their location, these antigens may be vaccine candidates as part of conjugate vaccines. Here, we describe the synthesis of extended oligomers of D-ribitol-1-phosphate linked to a spacer having an amino group at its terminus. The synthesis utilized a fully protected D-ribitol-phosphoramidite that was oligomerized in a stepwise fashion followed by deprotection. The free oligomers were connected to bovine serum albumin using oxime chemistry. Thus, the ribitol phosphate oligomers were converted into keto derivatives, and the albumin counterpart was decorated with aminooxy groups. Reaction of the functionalized saccharide and protein moieties afforded conjugates having up to 20 ribitol phosphate chains.

As polysialic acid (PSA), the capsule of Group B meningococcus (GBM) and Escherichia coli K1, is a component of mammalian glycopeptides, there is concern that vaccines against PSA could induce immunopathology. Purified PSA is not immunogenic; however, as a component of bacteria or bound to proteins, it induces protective antibodies. In this review, we did not unearth data indicating an association of IgG anti-PSA with immunopathology in experimental animals or humans. We found no increased incidence of autoimmunity from GBM infections in our review of the natural history/sequellae of Neisseria meningitis infections. Accordingly, we propose that clinical trials of PSA conjugate vaccines, be considered.

Neisseria meningitidisgroups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinantStaphylococcus aureusenterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives ofrSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs andrSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugate's immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately.

Replacement of cellular with acellular pertussis (aP) vaccines has considerably reduced the systemic reactions observed with diphtheria-tetanus toxoids-pertussis vaccine but has not eliminated the extensive swelling (sometimes involving an entire limb) observed after the fifth injection of diphtheria-tetanus toxoids-aP (DTaP) vaccine. This local reaction, which is likely an Arthus hypersensitivity reaction caused by high levels of antibodies reacting with DTaP vaccine, could discourage its use in adults, who serve as the major reservoir of pertussis for infants. That a critical level of antibodies to pertussis toxin is both essential and sufficient to prevent infection with Bordetella pertussis is derived from data from animal and clinical studies, including data showing the similarities between the immunity induced by diphtheria and pertussis toxoids. The genetically inactivated diphtheria and pertussis mutant toxins are more immunogenic and, therefore, induce comparable levels of antitoxin at lower protein levels than do the formalin-treated native toxins. Replacement of the diphtheria and aP components with these improved antigens will reduce the amount of protein in DTaP vaccine and, most likely, the incidence and severity of local reactions in teenagers and adults.

Using published data and the results of our studies, we hypothesized that a critical level of serum IgG antibodies to the surface structures of invasive pathogens (capsular polysaccharides of Haemophilus influenzae type b, pneumococcus, meningococcus, Salmonella typhi, Escherichia coli, and Staphylococcus aureus, the O-specific polysaccharide LPS domain of the LPS of Shigella, non-typhoidal Salmonella, and E. coli, and the capsular polypeptide of Bacillus anthraces) confer immunity to these pathogens. Covalent attachment to a protein increases their immunogenicity and bestows T-cell properties to these antigens. We have also shown that a critical level of serum IgG antibodies to pertussis toxin alone induces immunity on both an individual and on a community basis (herd immunity) to Bordetella pertussis. It is likely that all the above conjugates and pertussis toxoid will be incorporated into vaccines for routine infant immunization.

Bacillus pumilusstrain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide ofHaemophilus influenzaetype b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-α-galactopyranose (13%) and 2-acetamido-2-deoxy-α-glucopyranose (6%) on positionO-2, were found. A minor component was established to be a polymer of →3-O-(2-acetamido-2-deoxy-β-glucopyranosyl)-1→4-ribitol-1-OPO3→. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, includingH. influenzaetypes a and b and strains ofStaphylococcus aureusandStaphylococcus epidermidis.

Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule. The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development. In the present study we examined the prevalence and diversity of AM amongMycobacterium tuberculosisstrains using four AM-binding monoclonal antibodies (MAbs). One of these MAbs, MAb 9d8, is known to bind to AM specifically. By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8. However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11M. tuberculosisstrains tested, as demonstrated by capture ELISA. Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains. Mice immunized with an experimental AM-recombinantPseudomonas aeruginosaexoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge. Our results indicate that AM was detected in allM. tuberculosisstrains tested, with differences in epitope distributions of certain strains. In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.

Two major glycolipids, which comprise ≈36% of the total lipid mass from Borrelia burgdorferi , the etiological agent of Lyme disease, were investigated. We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position. The structures were identified as cholesteryl 6- O -acyl-β- d -galactopyranoside ( B. burgdorferi glycolipid 1, BbGL-I), and 1,2-di- O -acyl-3- O -α- d -galactopyranosyl- sn -glycerol (BbGL-II). The major fatty acids were palmitate and oleate. The structures were corroborated by gas–liquid chromatography MS, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling. This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria. Lipopolysaccharide was not detected in B. burgdorferi . The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure. BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.

This review summarizes the authors' studies in the past decade aimed at developing a synthetic oligosaccharide-based glycoconjugate vaccine to prevent a serious human disease caused by the Gram negative bacterium Shigelladysenteriae type 1. Starting from simple monosaccharides, oligosaccharides as large as a 24 monosaccharide-containing linear polymer, were assembled. Under suitable conditions, oligosaccharides containing 4 to 16 hexopyranose residues were covalently attached to an immunogenic protein. The serum response to the synthetic glycoconjugates depends, both on the size of the oligosaccharides, and on the molar ratio of the oligosaccharides to the carrier protein. Also reviewed are studies of the fine specificities of the interaction between oligosaccharides and anti-polysaccharide monoclonal antibodies as well as conformational studies of the synthetic oligosaccharides. 1 Polysaccharide-Based Vaccines 1.1 Surface Polysaccharides of Bacteria 1.2 Immunologic Properties of Polysaccharides 1.3 Polysaccharide-Protein Conjugates 1.3.1 Methods for the Conjugation of Polysaccharides to Proteins 1.3.2 Immunogenicity of Polysaccharide-Protein Conjugates 1.4 Synthetic Oligosaccharides Can be Superior to Natural Polysaccharides for Glycoconjugate Vaccines 1.5 Potentials of the O-Specific Polysaccharides of Shigellae for Vaccine Development 2 Chemical Synthesis of Oligosaccharides Related to the O-Specific Polysaccharide of S. dysenteriae Type 1 2.1 General Strategy 2.2 Synthesis of the Monosaccharide Building Blocks 2.2.1 The L-Rhamnose Moiety 2.2.2 The D-Galactose Moiety 2.2.3 The D-Glucosamine Synthons 2.3 Synthesis of a Complete Repeating Unit 2.4 Construction of Extended Oligosaccharides 2.5 The Lipophilic Protecting Group-Based Approach to Oligosaccharides 3 Covalent Attachment of the Oligosaccharides to Human Serum Albumin 3.1 Conjugation through a Secondary Spacer, Using Reductive Amination 3.2 Conjugation through Diels-Alder Cycloaddition Reactions 4 The Molecular Specificity of the Non-Covalent Binding between O-Specific Polysaccharide-Specific Antibodies and Oligosaccharides Related to the O-Specific Polysaccharide 5 Conformational Studies 6 Immunogenicity of the Protein Conjugates of the Synthetic Oligosaccharides in Mice 7 Conclusions and Future Prospects.

We measured anti-Haemophilus influenzae type a capsular polysaccharide serum immunoglobulin G antibodies in cord blood sera from Mexican (n = 68) and Chilean mothers (n = 72) by enzyme-linked immunosorbent assay. Measurable antibodies were found in 79.3% of samples. Immunoglobulin G antibodies correlated with serum bactericidal activity (r = 0.66). This enzyme-linked immunosorbent assay can be used for the evaluation of adaptive immune responses to Haemophilus influenzae type a and serosurveillance studies in populations at risk.

The outermost layer ofMycobacterium tuberculosisis composed primarily of two polysaccharides, glucan (GC) and arabinomannan. To analyze the surface polysaccharide composition ofM. tuberculosis, we generated a monoclonal antibody (MAb) that bindsM. tuberculosisGC and is known as MAb 24c5. Immunofluorescence and whole-mount immunoelectron microscopy indicated that GC is on the outermost portion of the bacteria.M. tuberculosisstrains Erdman and CDC 1551 were analyzed for their ability to bind MAb 24c5 after in vitro growth in media with and without the detergent Tween 80. MAb 24c5 bound to Erdman and CDC 1551 at all culture times with only slightly greater apparent affinity after extended culture in the absence of Tween 80, indicating that a stable amount of GC polysaccharide antigen is associated with the cell surface ofM. tuberculosis. An enzyme-linked immunosorbent assay indicated that GC is antigenically similar to glycogen, and the amount of GC antigen increased in the media ofM. tuberculosiscultures grown either with or without the detergent Tween 80. Other nontuberculosis mycobacteria have antigenically similar GCs on their surfaces after in vitro growth. Inoculation of mice with live bacilli but not inoculation with dead bacilli elicited a strong antibody response to GC consistent with production of this antigen in vivo. Our results provide a more comprehensive picture of theM. tuberculosiscell envelope and the conditions that allow expression ofM. tuberculosisGC.

To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis , a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis , and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis .

Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old.In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group.S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more.The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

The WHO strategy of instituting mass vaccination to combat meningococcal meningitis in sub-Saharan Africa has failed to control huge epidemics in 1996 and 1997. At best it would only prevent 50% of cases during the epidemic even under optimal conditions of detection and mobilization of personnel and resources. It also ignores the endemic prevalence of disease that would be categorized as epidemic in other parts of the world. In this respect this paper attempts to change the recommendation of the WHO for group A meningococcal meningitis. It recommends mass vaccination followed by routine vaccination and offers an agenda for the introduction of conjugate vaccines. Routine immunization with group A meningococcal polysaccharide has been noted to be effective. This approach serves as a means for rational development and field testing of the infrastructure for the deployment of a conjugate vaccine. The addition of meningococcal polysaccharide vaccines to routine immunization in African countries is recommended.

Unlike the native protein, a nontoxic peptide (repeating unit of the native toxin designated rARU) from Clostridium difficile toxin A (CDTA) afforded an antigen that could be bound covalently to the surface polysaccharides of pneumococcus type 14, Shigella flexneri type 2a, and Escherichia coli K1. The yields of these polysaccharide-protein conjugates were significantly increased by prior treatment of rARU with succinic anhydride. Conjugates, prepared with rARU or succinylated (rARUsucc), were administered to mice by a clinically relevant dosage and immunization scheme. All conjugates elicited high levels of serum immunoglobulin G both to the polysaccharides and to CDTA. Conjugate-induced anti-CDTA had neutralizing activity in vitro and protected mice challenged with CDTA, similar to the rARU alone. Conjugates prepared with succinylated rARU, therefore, have potential for serving both as effective carrier proteins for polysaccharides and for preventing enteric disease caused by C. difficile .

All acellular pertussis vaccines contain pertussis toxoid and induce protection against pertussis. This study investigated the relation between the postvaccination levels of pertussis toxin (PT) serum IgG and protection against pertussis. PT IgG was determined in sera obtained 21-77 days after the third vaccination from 813 children who received 3 doses of pertussis toxoid. The children were followed for 21-33 months after vaccination for the occurrence of pertussis. Of the children, 126 were exposed to pertussis in their households. The median PT IgG concentration was 79 U/mL in those who developed severe pertussis (>/=21 day of paroxysmal cough), 156 U/mL with mild pertussis (

Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)– Pseudomonas aeruginosa recombinant exoprotein A ( r EPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM 9 or r EPA, bound to the O-SP of Shigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM 9 served as a more immunogenic carrier than r EPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.

The age-related and T-cell independent properties of polysaccharides limit their use as vaccines. These limitations are overcome by covalently binding polysaccharides to proteins to form conjugates. Widespread use of Haemophilus influenzae type b (Hib) conjugates has virtually eliminated systemic infection caused by this pathogen, notably meningitis, in individuals of all ages. The principles derived from the development and use of Hib conjugates have been applied to other capsulated pathogens including pneumococci and meningococci. We have shown that vaccine-induced serum IgG antibodies to the surface polysaccharides of enteric pathogens confer immunity to typhoid fever (Vi) and to S. sonnei. Our preliminary data show that synthetic saccharides provide a method for increasing the immunogenicity of conjugates and permitted more direct characterization of a S. dysenteriae type 1 conjugate. Both the chain length and density of the saccharides on the protein were related to the immunogenicity of conjugates in mice. A synthetic approach has also been extended to the LPS types of Vibrio cholerae O1.

Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain ofShigellalipopolysaccharide (LPS) confers immunity. The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies. The O-SP ofShigella dysenteriaetype 1 (≈27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 1.0. Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24. Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA. Outbred 6-week-old female mice were injected s.c. three times at biweekly intervals with 2.5 μg of saccharide as a conjugate and were bled 7 days after the second and third injections. Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P= 0.0001). The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (15.5 ELISA units) followed by the octamer with 20 chains (11.1 ELISA units) and the dodecamer with 10 chains (9.52 ELISA units). Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned.

Escherichia coli O148 is a non-encapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit 3)-α-d-Rhap-(1→2)-α-d-Glcp-(1→3)-α-d-GlcNAcp-(1→3)-α-d-Rhap-(1→ differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a d-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SP's of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPS's of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-PS's that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SP's of both bacteria.

A phase 1 study of a recombinant mutant protective antigen (rPA) vaccine was conducted in 186 healthy adults aged 18 to 45 years. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-μg dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. Vaccinees were examined at the clinic once following each injection, at 48 to 72 h postinjection. Adverse reactions were recorded in diaries for 7 days. Sera were collected before each injection and 1 week after the 1st, 2 weeks after the 3rd and 4th, and 1 year after the 4th. Serum anti-PA IgG was assayed by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). All formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 μg/ml). The lowest levels were induced by the formalin-only-treated rPA; there was no statistical difference between levels induced by alum-adsorbed and formalin-treated/alum-adsorbed rPA or by the two dosages. The antibody levels declined in all groups during the 1-year intervals after the 3rd and 4th injections but less so during the 2nd year, after the 4th injection (fold decreases were 10 to 25 versus 3.4 to 7.0, P < 0.001). There were too few AVA recipients for statistical comparisons, but their antibody levels followed those of rPA. Anti-rPA measured by ELISA correlated with TNA titers ( r = 0.97). These data support studying alum-adsorbed rPA in children.

Background Most early-onset group B streptococcal (GBS) disease in recent years has occurred in newborns of prenatally GBS-negative mothers who missed intrapartum antibiotic prophylaxis (IAP). We aimed to assess the accuracy of prenatal culture in predicting GBS carriage during labor, the IAP use, and occurrence of early-onset GBS disease. Methods We obtained vaginal-rectal swabs at labor for GBS culture from 5497 women of ≥ 32 weeks' gestation and surface cultures at birth from newborns between February 5, 2008 and February 4, 2009 at 3 hospitals in Houston, TX and Oakland, CA. Prenatal cultures were performed by a healthcare provider during routine care, and culture results were obtained from medical records. The accuracy of prenatal culture in predicting intrapartum GBS carriage was assessed by positive and negative predictive values. Mother-to-newborn transmission of GBS was assessed. Newborns were monitored for early-onset GBS disease. Results GBS carriage was 24.5% by prenatal and 18.8% by labor cultures. Comparing prenatal with labor GBS cultures of 4696 women, the positive predictive value was 50.5% and negative predictive value was 91.7%. IAP, administered to 93.3% of prenatally GBS-positive women, was 83.7% effective in preventing newborn's GBS colonization. Mother-to-newborn transmission of GBS occurred in 2.6% of elective cesarean deliveries. Two newborns developed early-onset GBS disease (0.36/1000 births); the prenatal GBS culture of one was negative, the other's was unknown. Conclusions IAP was effective in interrupting mother-to-newborn transmission of GBS. However, approximately 10% of prenatally GBS-negative women were positive during labor and missed IAP, whereas approximately 50% of prenatally GBS-positive women were negative during labor and received IAP. These findings emphasize the need for rapid diagnostics during labor.

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti- B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis . Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8–17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis , and the titers correlated with ELISA-measured antibody levels ( r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.

Borrelia burgdorferi is the etiological agent for Lyme disease (LD), the most common vector borne disease in the United States. There is no human vaccine against LD currently available. Our approach to a vaccine is based on its surface-exposed glycolipids. One group of these glycolipids termed BBGL-2 consists of 1,2-di-O-acyl-3-O-(α-d-galactopyranosyl)-sn-glycerol congeners having palmitic, oleic, stearic, linoleic, and myristic acids. In order to delineate the immunodominant region(s) of the BBGL-2 components, we embarked on a synthetic project to provide available structurally defined, homogeneous analogs of BBGL-2 that might help identify the best vaccine candidate. The antigenicity of the synthetic glycolipids was examined by dot-blot analysis using mice sera obtained by immunization with killed B. burgdorferi cells, with native BBGL-2 in complete Freund's adjuvant, as well as sera obtained from patients with Lyme disease. We found that the presence of two acyl groups in the glycerol moiety was essential for antigenicity. At least one of these groups must be an oleoyl moiety. Neither the anomeric configuration of the galactose nor the configuration of the glycerol at C-2 was a decisive factor. Based on these findings we designed an 'unnatural' BBGL-2 analog having the structure 3-O-(β-d-galactopyranosyl)-1,2-di-O-oleoyl-dl-glycerol which is easier and less expensive to synthesize than the other BBGL-2 congeners prepared in this study. This substance proved to be antigenic and is considered a candidate vaccine for Lyme disease.

Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussis were measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.

Haemophilus influenzae type a (Hia) is an important pathogen for some American Indian, Alaskan native, and Northern Canada aboriginal populations. Assays to measure serum bactericidal activity (SBA) to Hia have not been developed or validated. Here, we describe two methods for the measurement of SBA: SBA with a viability endpoint (CFU counts) and SBA with a fluorometric endpoint using alamarBlue as the metabolic indicator. Both SBA assays measure Hia-specific functional antibody and correlate with anti-Hia IgG enzyme-linked immunosorbent assay (ELISA) concentration of naturally acquired antibodies.

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Herein, we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have d-configuration except l-Rha)

There is yet no licensed vaccine against malaria, a serious human disease affecting mostly children, with an annual death rate of about one million. Plasmodia , the malaria-causing parasites, have two obligatory hosts: mammals or birds, in which they multiply asexually, and mosquitoes with sexual multiplication. The most common and serious type of malaria is caused by Plasmodium falciparum . The circumsporozoite protein (CSP), a major surface antigen of sporozoites, is a protective antigen. A unique feature of P. falciparum CSP is its large central domain composed of over 30 tetrapeptide repeats of Asn-Ala-Asn-Pro (NANP). Several NANP peptide-protein conjugates were tested clinically but elicited a low level of CSP antibodies for a short duration. To provide a CSP-based candidate vaccine, we investigated recombinant CSP and NANP conjugates of various peptide lengths, with different N-terminal amino acids, bound at different ratios to various carrier proteins. Injected into mice, CSP alone and CSP or NANP conjugates induced antibodies with booster responses and were positive by the sporozoite imunofluorescent assay. The use of the mosquito stage P. falciparum ookinete surface protein, Pfs25, cross-linked onto itself as a carrier for NANP, induced in mice high levels of uniquely long-lasting antibodies to both vaccine components with secondary biological activities, that will provide immunity to liver infection by sporozoites and block transmission by mosquitoes.