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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.

Stereocontrolled, stepwise synthesis of decyl glycosides of α-(1 → 2)-linked di- to pentaglucosides (1–5) is described; these constitute fragments of Polysaccharide II of Mycobacterium tuberculosis. Phenyl 3,4,6- tri -O- acetyl -2-O- benzyl-1-thio-α- d -glucopyranoside (7) was used as the single key intermediate, obtained from 1,3,4,6- tetra -O- acetyl -2-O- benzyl-β- d -glucopyranose (6) and PhSSiMe3. Halogenolysis of 7 afforded the isolated β bromide (10) and β chloride (13). Solvolysis of 10 with decanol without heavy metal salts gave decyl 3,4,6- tri -O- acetyl -2-O- benzyl-α- d -glucopyranoside (14) in a highly stereoselective reaction, in high yield. Subsequent, iterative hydrogenolytic removal of the O-benzyl group and glycosylation with the β-chloride 13 under catalysis by silver salts afforded the protected di- to penta-saccharide glycosides 16, 19, 21, and 23, which were conventionally deblocked.

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.

A glycosyl trichloroacetimidate derivative ( 1 ) of the tetrasaccharide α- d -Gal p -(1→3)-α- d -Glc p NAc-(1→3)-α-L-Rha p -(1→3)-α-L-Rha p was synthesized in a highly stereoselective, stepwise manner, using methyl 1-thioglycosides of L-rhamnose, 2-azido-2-deoxy-D-glucose and D-galactose, as major intermediates. The protecting group scenario in compound 1 permits regioselective deblocking at its “non-reducing end” unit. Therefore 1 is a suitable intermediate for the preparation of extended fragments of the title polysaccharide.

The antibody responses of BALB/c mice to serotype A Cryptococcus neoformans capsular polysaccharide (CNPS) were compared after cryptococcal infection and immunization with a serotype A glucuronoxylomannan-tetanus toxoid conjugate (GXM-TT). Infection rarely resulted in a rise of serum antibody titer to CNPS. In contrast, mice immunized with GXM-TT produced serum IgM and IgG to CNPS. Six IgM and one IgG1 monoclonal antibodies (MAbs) were generated from the spleen of one infected mouse. Nine IgM, 1 IgG3, 16 IgG1, and 7 IgA MAbs were generated from the spleen of one GXM-TT-immunized mouse. All MAbs generated from both mice bound to the GXM fraction of the capsular polysaccharide. For some MAbs, de-O-acetylation of serotype A GXM abolished or greatly reduced MAb binding compared with the native GXM. All MAbs reacted with CNPS from C. neoformans serotypes A-D. MAbs generated from the infected mouse competitively inhibited the binding of MAbs generated from the GXM-TT-immunized mouse. These results indicate that some antibodies elicited by infection with C. neoformans or by immunization with GXM-TT bind to the same antigenic determinant in the GXM.

The chemical synthesis of a linear octasaccharide, composed of two contiguous repeating units of the O-specific determinant of Shigella dysenteriae type 1 is described. 1H-NMR data indicate that this octasaccharide possesses conformational features of the native polysaccharide.

Background Despite its high worldwide morbidity and mortality, there is yet no licensed vaccine for shigellosis. We reported the safety and immunogenicity of Shigella O-specific polysaccharide–protein conjugates in adults and young children and efficacy of Shigella sonnei conjugate in young adults. Methods A double-blinded, randomized and vaccine-controlled Phase 3 evaluation of S. sonnei and Shigella flexneri 2a O-SP–rEPA conjugates, 25 μg, injected IM twice, 6 weeks apart, into healthy 1–4 years old, is reported. The children were followed for 2 years by telephone every other week and stool cultures were obtained for each episode of acute diarrhea (≥3 loose stools/day or a bloody/mucous stool). Sera were taken randomly from 10% of the participants for IgG anti-LPS and anti-carrier levels. Results Of the 2799 enrollees, 1433 received S. sonnei and 1366 S. flexneri 2a conjugates; 2699 (96.4%) completed the 2-year follow-up. Local reactions occurred in ∼5% and ∼4% had temperatures ≥38.0 °C lasting 1–2 days. There were no serious adverse events attributable to the vaccines. Of the 3295 stool cultures obtained, 125 yielded S. sonnei and 21 S. flexneri 2a. Immunogenicity and efficacy were age-related. The overall efficacy of the S. sonnei conjugate was 27.5%; 71.1% ( P = 0.043) in the 3–4 years old. The numbers for S. flexneri 2a were too few for meaningful analysis. Cross-protection by S. flexneri 2a for non-vaccine S. flexneri types was found, but the numbers were too few for statistical significance. There was an age-related rise of vaccine-specific IgG anti-LPS in both groups, peaking at about 10 weeks and declining thereafter, but remaining ≥4-fold higher than in the controls 2 years after the second dose. Conclusions Shigella conjugates are safe and immunogenic in 1–4 years old. The S. sonnei conjugate elicited 71.1% efficacy in the 3–4 years old and can be predicted to be efficacious in individuals older than 3 years of age. These results urge studies with our improved conjugates.

Production of influenza vaccines requires a minimum of 6 months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni(+)-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines two or three times 2 weeks apart. Sera were collected 1 week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (GM 449EU) was elicited by three injections of 15microg alum-adsorbed rHA. Dosages of 5microg of rHA formulated with formalin and alum, and 5microg alum-adsorbed rHA elicited IgG anti-HA of GM 212 and 177EU, respectively. HI titers, >or=40 were obtained in >or=80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in 3-4 weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.

A critical level of serum IgG pertussis toxin antibody is both essential and sufficient to confer individual and herd immunity to pertussis. Monocomponent pertussis toxoid conferred such immunity in Sweden and in Denmark. We refute the notion that filamentous hemagglutinin, pertactin, and fimbriae add to the immunity conferred by pertussis toxoid and describe the artifact created when efficacy is estimated for multicomponent pertussis vaccines. Lastly, the genetically-inactivated mutant pertussis toxoid is safer, more immunogenic, and should be more effective than the current chemically-inactivated pertussis toxin.

Bacillus anthracis is a spore-forming bacterium that causes anthrax in humans and in other mammals. The glycoprotein BclA ( Bacillus collagen-like protein of anthracis ) is a major constituent of the exosporium, the outermost surface of B. anthracis spores. The glycosyl part of BclA is an oligosaccharide composed of 2- O -methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy- d -glucose, referred to as anthrose, and three rhamnose residues. A structure similar to anthrose, 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy- d -glucose is found in the side chain of the capsular polysaccharide (CPS) of Shewanella spp. MR-4. Under certain growth conditions the bacteria produce a variant CPS lacking one methyl group on the hydroxybutyrate, 4-(3-hydroxybutanamido)-4,6-dideoxy- d -glucose. Contrary to anthrose, neither of the Shewanella CPSs is 2- O methylated. Here, we report that both Shewanella CPS variants react with anti- B. anthracis spore sera. We also found that these antisera reacted with flagellae of Pseudomonas syringae , reported to be glycosylated with a similar terminal saccharide, 4-(3-hydroxybutanamido)-4,6-dideoxy-2- O -methyl- d -glucose. Sera produced by immunization with Shewanella or P. syringae cells bound to B. anthracis spores but not to Bacillus cereus spores in a fluorescent microscopy assay. These experiments show that methylation of the anthrose at the O- 2 of the sugar ring and at the C -3 of 3-hydroxybutyrate are not essential for induction of cross-reactive antibodies. We report the preparation, characterization, and antibody responses to protein conjugates of the two variants of Shewanella CPS. Both conjugates induced antibodies that bound to both Shewanella CPS variants by ELISA and to B. anthracis spores, as detected by fluorescent microscopy. We propose the use of Shewanella CPS conjugates as a component of an anthrax vaccine.

Bordetellae are Gram-negative bacilli causing respiratory tract infections of mammals and birds. Clinically important are B. pertussis, B. parapertussis and B. bronchiseptica. B. pertussis vaccines have been successful in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action are not established. There is no vaccine against B. parapertussis. Based on the concept that immunity to non-capsulated Gram-negative bacteria may be conferred by serum IgG anti-LPS we studied chemical, serological and immunological properties of the O-specific polysaccharides (O-SP) of B. bronchiseptica and B. parapertussis obtained by different degradation procedures. One type of the B. parapertussis and two types of B. bronchiseptica O-SP were recognized based on the structure of their non-reducing end saccharide; no cross-reaction between the two B. bronchiseptica types was observed. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SP. Conjugates of B. bronchiseptica and B. parapertussis O-SP were prepared by two methods: using the anhydro-Kdo residue exposed by mild acid hydrolysis of the LPS or the 2,5-anhydromannose residue exposed by deamination of the core glucosamine of the LPS, for binding to an aminooxylated protein. Both coupling methods were carried out at a neutral pH, room temperature, and in a short time. All conjugates, injected as saline solutions at a fraction of an estimated human dose, induced antibodies in mice to the homologous O-SP. These methodologies can be applied to prepare O-SP-based vaccines against other Gram-negative bacteria.

Contributions to science and to the practice of medicine are built on the shoulders of our predecessors. For us, the shoulders of Dr. Maxwell Finland were singularly broad and strong: In addition to his skills as a physician and a teacher, he was a visionary and a scientist. Dr. Finland started his career by caring for and studying patients with pneumooccal pneumonia [1,2]. He realized the need for understanding the immunologic properties of the pneumococcus capsular polysaccharides (CP) and their relation to clinical disease caused by this pathogen. Some of his contributions that guided our efforts to develop effective vaccines for prevention of systemic infections due to capsulated bacterial pathogens are cited in table 1 [3-16]. Our main interest has been toward preventing meningitis and other systemic infections caused by Haemophilus influenzae type b (Hib). Progress by us and by other laboratories toward developing Hib conjugates has been rapid, and vaccines manufactured by three companies have been licensed recently for children ^18 months of age. The advent of Hib conjugates licensed for universal immunization of infants, administered concurrently with diphtheria and tetanus toxoids and pertussis vaccine (DTP), is imminent. We are confident that chemists and engineers will continue to improve these first Hib conjugates and that even more effective products will be available soon. Accordingly, in addition to reviewing our contributions and those of our colleagues with these new Hib vaccines, we will illustrate how the development of polysaccharide-protein conjugates may provide opportunities for studying the pathogenesis of other bacterial diseases and ultimately for preventing them through new vaccines. Surface polysaccharides may serve both as virulence factors and as protective antigens for bacteria whose invasion of the blood is their primary pathogenic event [17]. These

Pneumococcus vaccine, injected alone or mixed with diphtheria-tetanus toxoid-pertussis, did not elicit significant concentrations of pneumococcus type 6 antibodies in 2- to 5-year-old sickle cell anemia patients (n = 22). Reinjection 5 months later failed to elicit a booster response to pneumococcus type 6. We then injected conjugates of pneumococcus type 6B and of Haemophilus influenzae type b (Hib), each bound to tetanus toxoid (TT), alternatively at monthly intervals into sickle cell anemia patients of the same age group (n = 25); most received 3 injections of each vaccine. Pneumococcus vaccine was administered to 19 patients and Hib to 1 at approximately 1 year of age. Blood samples were taken before each and approximately 6 months after the last injection. Infrequent and minimal local reactions and only 6 episodes of fever (3%) occurred after injection of the conjugates. Pneumococcus type 6B-TT elicited a rise in the geometric mean concentration of pneumococcus type 6 antibodies (Ab) from 104 ng of antibody nitrogen (AbN)/ml in preimmunization sera to 385 ng of AbN/ml after the first injection (P less than 0.01). There were further increases after the 2 subsequent injections; 6 months after the third injection, the mean concentration was 940 ng of AbN/ml and 15 of 16 (94%) had greater than 300 ng of AbN/ml. Hib-TT elicited a 160-fold increase of Hib antibodies to a geometric mean concentration of 39.0 micrograms of Ab/ml after the first injection. These levels rose approximately 2-fold following 2 additional injections to 71.7 micrograms/ml and declined to 10.7 micrograms/ml at the 6-month sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemophilus influenzae was first described by Pfeiffer in 1892 ([30][1]). Forty years later Pittman identified six capsular polysaccharides (types) of H. influenzae (a, b, c, d, e, and f) whose structures were later elucidated (Table [1][2]) ([12][3], [31][4], [40][5]). Systemic infections in

Malaria is a leading cause of morbidity and mortality, estimated to cause >1 million childhood deaths annually.Plasmodium falciparumcauses the most severe form of the disease. There is as yet no licensed vaccine for this disease, despite over a half century of research. In this study, we investigated a transmission-blocking vaccine candidate, the ookinete surface protein Pfs25. Antibodies against Pfs25, drawn in during a bite, can block parasite development in the mosquito midgut, preventing transmission to other individuals. Pfs25 is a low-molecular-weight protein, by itself not immunogenic. To increase its immunogenicity, we investigated several methods of conjugating Pfs25 to itself and to other proteins: recombinantPseudomonas aeruginosaexotoxin A, and ovalbumin, using amide, hydrazone, or thioether linkages. All conjugates were immunogenic and induced booster responses in mice. The scheme to form amide bonds between proteins by using adipic acid dihydrizide as a linker produced the most immunogenic conjugates. Adsorption of the conjugates onto aluminum hydroxide further increased the antibody response. Remarkably, the antibody levels 3 or 7 months after the last injection were significantly higher than those 1 wk after that injection. The observed transmission-blocking activity of immune sera correlated with antibody levels measured by ELISA.

The capsule of Bacillus anthracis , composed of poly-γ- d -glutamic acid (γDPGA), is an essential virulence factor of B. anthracis . The capsule inhibits innate host defense through its antiphagocytic action. γDPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic γDPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between γDPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal γDPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol γDPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 μg of γDPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-γDPGA levels.

IgG antibodies against Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) and tetanus toxoid (TT) were measured for 53 children, 10 years of age, before and 1 month after a booster dose of diphtheria-tetanus vaccine (DT). All children had been vaccinated at 3, 5 and 12 months of age with DT and a Hib-TT conjugate. Geometric mean concentrations of Hib CPS serum IgG antibody were 4.16 and 4.30 microg/mL before and after the DT booster, respectively. The geometric mean concentration of TT IgG antibody increased from 0.09 IU/mL to 4.58 IU/mL (P < 0.001). Hib CPS IgG levels remained well above protective titers for 9 years after 3 doses of Hib-TT appropriately spaced in infancy. A booster dose of TT did not affect Hib CPS antibody concentrations but induced a pronounced IgG response against TT.

In a double-blind, randomized, and placebo-controlled previous trial, the efficacy of Vi- r EPA for typhoid fever in 2- to 5-year-olds was 89.0% for 46 months. Vi- r EPA contained 25 μg of Vi and induced a greater-than-eightfold rise in immunoglobulin G (IgG) anti-Vi in all of the vaccinees tested. In this investigation, we conducted a dosage-immunogenicity study of 5, 12.5, and 25 μg of Vi- r EPA in this age group. Two doses of Vi- r EPA were injected 6 weeks apart. Blood samples were taken before and at 10 weeks (4 weeks after the second injection) and 1 year later. All postimmunization geometric mean (GM) levels were higher than the preimmune levels ( P < 0.0001). At 10 weeks, the GM IgG anti-Vi level elicited by 25 μg (102 EU/ml) was higher than those elicited by 12.5 μg (74.7 EU/ml) and 5 μg (43 EU/ml) ( P < 0.004): all of the children had ≥3.52 EU/ml (estimated minimum protective level). One year later, the levels declined about sevenfold (13.3 and 11.3 versus 6.43 EU/ml, P < 0.0001) but remained significantly higher than the preimmune levels ( P < 0.0001), and >96% of the children had a greater-than-eightfold rise. This study also confirmed the safety and consistent immunogenicity of the four lots of Vi- r EPA used in this and previous trials.

Background Staphylococcus aureus , the first or second most common pathogen isolated from patients, is capsulated; there are at least 12 capsular types, and types 5 and 8 comprise approximately 85% of blood. Types 5 and 8, composed of a trisaccharide repeat unit including a mannose uronic acid and 2 fucoses, are non-immunogenic. As protein conjugates, they induce opsonophagoctyic antibodies that confer type-specific active and passive protection in mice. Methods A phase II study of patients with end-stage renal disease showed that these conjugates induced approximately one third of the immunoglobulin G antibody of healthy individuals. Increasing the dose to 100 μg of polysaccharide induced levels similar to that in healthy individuals injected with 25 μg. Results In a double-blinded randomized and controlled study of patients undergoing renal dialysis, the conjugates induced statistically significant protection against bacteremia for as long as 10 months after immunization. The estimated protective level was 80 μg Ab/mL. At re-injection approximately 2 years later, 83 of 83 recipients responded with protective levels. Conclusions Conjugate vaccine-induced antibodies to the types 5 and 8 capsular polysaccharide antibodies of S aureus prevent bacteremia caused by this pathogen. The extent and duration of conjugate-induced immunity can be extended by re-immunization approximately 1 year later. Studies of patients undergoing cardiovascular surgery who would be immunized with the staphylococcus conjugates when they are immunologically intact are planned.

Shigella conjugate vaccines have been shown to be safe, immunogenic and efficacious in adult volunteers. We have now investigated the safety and immunogenicity of investigational Shigella sonnei and Shigella flexneri 2a conjugate vaccines in 1- to 4-year-old children, the age group at greatest risk for shigellosis.The O-specific polysaccharides of S. sonnei and S. flexneri 2a, the two most common shigellae from patients in Israel, were bound to medically useful carrier proteins to form conjugates. Eighty healthy 1- to 4-year-olds were randomized to receive two 0.5-ml im injections 6 weeks apart of either S. sonnei-CRM(9) or S. flexneri 2a-rEPA(succ). Blood was taken before, 6 weeks after the first injection, 4 weeks after the second injections and 2 years after immunization for assay of IgG anti-lipopolysaccharide, diphtheria toxin and Pseudomonas aeruginosa exotoxin A antibodies by enzyme-linked immunosorbent assay.During an 8-day surveillance period after each immunization, low fever (37.8-39.0 degrees C) lasting only 24 to 48 h occurred in 2 of 40 recipients after the first injection and 4 of 40 recipients after the second injection of S. flexneri 2a-rEPA(succ) and in 2 of 38 of S. sonnei-CRM(9) after the second injection; no fever was detected after the first injection. Liver function tests were normal in all vaccinees. S. sonnei-CRM(9) elicited a4-fold rise in IgG anti-LPS in 92.1% and S. flexneri 2a-rEPA(succ) in 85% (P0.0001) after the second injection; both conjugates elicited type-specific booster responses. At 2 years the geometric mean concentrations of both IgG anti-lipopolysaccharides were significantly higher than preimmunization levels. A4-fold rise of IgG anti-diphtheria (65.8%) and IgG anti-ETA (77.5%) was observed.These experimental Shigella conjugate vaccines were safe and immunogenic in 1- to 4-year-old children.

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.

Both the protective antigen (PA) and the poly(γ-d-glutamic acid) capsule (γdPGA) are essential for the virulence ofBacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-γdPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic γdPGA or corresponding synthetic peptides were bound to BSA, recombinantB. anthracisPA (rPA), or recombinantPseudomonas aeruginosaexotoxin A (rEPA). To identify the optimal construct, conjugates ofB. anthracisγdPGA,Bacillus pumilusγdLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of thedorlconfiguration with active groups at the N or C termini, were bound at 5–32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-γdPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-γdPGA were elicited by decamers of γdPGA at 10 –20 mol per protein bound to the N- or C-terminal end. High IgG anti-γdPGA levels were elicited by two injections of 2.5 μg of γdPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies.rPA was the most effective carrier. Anti-γdPGA induced opsonophagocytic killing ofB. anthracistox–, cap+. γdPGA conjugates may enhance the protection conferred by PA alone. γdPGA-rPA conjugates induced both anti-PA and anti-γdPGA.

Nontypeable Haemophilus influenzae (NTHi) accounts for about one-third of purulent otitis media (OM) in children and is a common cause of pulmonary infection in adults with decreased resistance. Based upon sero-epidemiological data in humans and immunochemical data in laboratory animals, a lipooligosaccharide (LOS)–tetanus toxoid (TT) conjugate was prepared and evaluated for its safety and immunogenicity in a Phase I study of 40 healthy adults. The conjugate was injected intramuscularly into all volunteers: 28 of them received a second injection 14 weeks later. Local and systemic reactions were monitored and sera, taken before and 2, 6, 14, 16, and 38 weeks after injection, were assayed for IgG, IgA, and IgM antibodies to the LOS by ELISA and for bactericidal activity. The results indicate that there were no significant local or systemic reactions after either injection. All volunteers had pre-existing IgG anti-LOS. The geometric mean (GM) level rose from 14 to 40 at 2 weeks, remained at 35 at 6 weeks (40 or 35 versus 14, P

Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinosecontaining carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human VH determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and VH determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection. Tuberculosis (TB) is a leading cause of mortality worldwide, and it is estimated that 1.86 billion people are infected with Mycobacterium tuberculosis (13). Contributing factors to this severe health problem are the human immunodeficiency virus epidemic, the long and complicated course of treatment, drug resistance, and a lack of efficient diagnostic and preventive modalities. In addition, we lack a full understanding of the immunopathogenesis of TB. Our group is interested in the M. tuberculosis carbohydrate arabinomannan (AM), which is thought to be a component of the mycobacterial capsule. This interest arose from the generation of a monoclonal antibody (MAb), 9d8, that prolonged the survival of mice infected with M. tuberculosis (36) and binds AM. These findings suggested

In patients with decreased resistance to infection, Staphylococcus aureus is a major cause of bacteremia and its complications. The capsular polysaccharides are essential for the pathogenesis of and immunity to S. aureus infection and are targets for vaccines.In a double-blind trial involving patients with end-stage renal disease who were receiving hemodialysis, we evaluated the safety, immunogenicity, and efficacy of a vaccine with S. aureus type 5 and 8 capsular polysaccharides conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A. Between April 1998 and August 1999, 1804 adult patients at 73 hemodialysis centers were randomly assigned to receive a single intramuscular injection of either vaccine or saline. IgG antibodies to S. aureus type 5 and 8 capsular polysaccharides were measured for up to two years, and episodes of S. aureus bacteremia were recorded. Efficacy was estimated by comparing the incidence of S. aureus bacteremia in the patients who received the vaccine with the incidence in the control patients.Reactions to the vaccine were generally mild to moderate, and most resolved within two days. The capsular polysaccharides elicited an antibody response of at least 80 microg per milliliter (the estimated minimal level conferring protection) in 80 percent of patients for type 5 and in 75 percent of patients for type 8. The efficacy during weeks 3 to 54 was only 26 percent (P=0.23). However, between weeks 3 and 40 after vaccination, S. aureus bacteremia developed in 11 of 892 patients in the vaccine group who could be evaluated for bacteremia, as compared with 26 of 906 patients in the control group (estimate of efficacy, 57 percent; 95 percent confidence interval, 10 to 81 percent; nominal P=0.02).In patients receiving hemodialysis, a conjugate vaccine can confer partial immunity against S. aureus bacteremia for approximately 40 weeks, after which protection wanes as antibody levels decrease.

Pertussis re-emerged in Sweden with a cumulative incidence of about 60% during the first 10 years of life, when the locally produced cellular vaccine lost its efficacy around 1970 and general vaccination was discontinued in 1979. The epidemiology, clinical features, and immunology of pertussis and a monocomponent pertussis toxoid vaccine were studied in Göteborg, Sweden.After phase 1 and 2 studies, a randomized, double-blind, placebo-controlled trial of pertussis toxoid (PTox), compounded with diphtheria and tetanus toxoids, was administered to 3450 children according to the Swedish schedule at 3, 5, and 12 months of age. After a mean follow-up of 18 months, the efficacy was 71% overall and 75% in household contacts, respectively. A statistically significant correlation was found between the level of PTox-induced antibodies and protection against pertussis. As observed with cellular and with multicomponent acellular vaccines, PTox reduced the severity of disease and the percent of children with positive cultures. Furthermore, vaccination reduced the transmission of Bordetella pertussis to household contacts in the vaccinees compared with the controls who received only diphtheria and tetanus toxoids. Patients with culture-verified Bordetella parapertussis infection reacted with antibodies to pertactin and to filamentous hemagglutinin but not to pertussis toxin, and some subsequently developed pertussis. The antibody responses of patients with pertussis to the surface polysaccharides of B pertussis and to B parapertussis were cross-reactive serologically. Serosurveys showed that only antibodies to pertussis toxin were related to the occurrence of pertussis in the general population: antibodies to filamentous hemagglutinin and pertactin were probably stimulated by antigens of other bacteria as well as Bordetellae.Mass vaccination of Göteborg children born in the 1990s was started in 1995. In February 1999, about 55% had been vaccinated and both B pertussis and pertussis decreased significantly in individuals of all ages (herd immunity). Similar to diphtheria, PTox-induced immunity to pertussis occurs both on an individual and community basis.The apparent greater efficacy of multicomponent acellular pertussis vaccines compared with monocomponent PTox was proposed to be an artifact created when the diagnosis of pertussis was made by the serologic criteria of the World Health Organization only. Our conclusion is that PTox is both an essential and alone sufficient antigen in acellular pertussis vaccines.

The outermost layer ofMycobacterium tuberculosiscontains two major polysaccharides, arabinomannan (AM) and glucan (GC). We studied the in vitro and in vivo expression of anM. tuberculosisAM antigen using monoclonal antibody (MAb) 9d8 (2a), an isotype-switched variant of the immunoglobulin G3 (IgG3) MAb 9d8. MAb 9d8 had been previously shown to bindM. tuberculosisAM and theM. tuberculosissurface. Our in vitro experiments showed that MAb 9d8(2a) bound strongly to whole-cellM. tuberculosisErdman but not to the CDC 1551 strain grown in medium for an extended period. However, AM antigen was detected in the culture supernatant of both strains, and its concentration increased in a time-dependent manner. The detection of AM antigen from both strains was decreased in the presence of Tween 80. In mice infected withM. tuberculosisErdman, AM antigen accumulated in organ homogenates concomitant to an increase in bacterial organ burden and an increase in IgG and IgM titer to AM. These results (i) indicate that the surface expression of AM during in vitro growth changes with culture age, is strain dependent, and is affected by the presence of Tween 80 in the culture media; (ii) show that AM is produced by bacteria growth in vivo; and (iii) demonstrate that the amount of in vivo-detected AM can be dependent on the number of bacteria in the infected organ.

Shigellosis remains a serious and common disease (16, 19, 23, 26, 28, 38, 46, 55, 56). In addition to causing watery diarrhea, shigellae are a major cause of dysentery (fever, cramps, and blood and/or mucus in the stool) (19, 46, 49, 55, 56). Not commonly appreciated is that dysentery, not watery diarrhea, retards growth in children (7, 9, 19, 30, 38, 46, 49, 55, 56). Although Shigella dysenteriae type 1 was discovered as the cause of epidemic dysentery in Japan in 1898 (53), there is neither a licensed vaccine for it nor a consensus as to the mechanism(s) of host immunity to Shigella (11, 18, 31, 38, 46, 47). Vaccine development has been hampered by three factors: (i) the ineffectiveness of parenterally injected inactivated whole-cell vaccines which led to the belief that serum antibodies do not confer immunity (25, 32); (ii) the lack of a suitable animal model (46); and (iii) only indirect evidence of immune mechanism(s) in humans (11, 14, 38, 46–48). The O-specific polysaccharide (O-SP) domain of lipopolysaccharide (LPS) is both an essential virulence factor and a protective antigen of Shigella (46). Convalescence from shigellosis confers LPS-specific immunity, although incomplete and of limited duration (6, 18, 31, 38, 46). The following data indicate serum immunoglobulin G (IgG) anti-O-SP confers immunity to shigellosis. (i) Correlation was found between the level of IgG LPS antibodies and resistance to shigellosis among Israeli solders (11, 14). (ii) There is an inverse relationship between the age incidence of shigellosis and the presence of IgG antibodies to the LPS of Shigella (41, 46). The peak incidence of shigellosis is in children and young adults; the disease is rare in infants and in older adults (14, 15, 20, 21, 23, 26, 28, 38, 46). Most newborns and adults have serum LPS antibodies that may be stimulated by cross-reacting bacteria (46–48). (iii) In a double-blind, vaccine-controlled randomized trial of our S. sonnei-rEPA (Pseudomonas aeruginosa recombinant mutant exoprotein A) conjugate (15), 1,447 Israel Defense Force (IDF) recruits from seven companies at separate field sites were vaccinated with S. sonnei-rEPA or one of two control vaccines. Shigellosis occurred in three units 2 to 3 months after vaccination; S. sonnei-rEPA induced an overall efficacy of 74% (P = 0.001). In one company, infection with S. sonnei occurred within 1 to 17 days of injection. Nevertheless, S. sonnei-rEPA conferred 43% (P = 0.04) protection, suggesting that our conjugates may be of value when administered during epidemics. A correlation was demonstrated only between the level of IgG anti-LPS and protection (15). Since serum antibodies are the main, if not the only, host mechanism induced by polysaccharide-protein conjugates, these data provide evidence that a critical level of IgG anti-LPS confers immunity to shigellosis (11, 46–48). The immunogenicity of polysaccharide-based vaccines, including conjugates, is age dependent (46–48). We improved the immunogenicity of Shigella conjugates as assayed in mice by introduction of another carrier protein, Corynebacterium diphtheriae CRM9 (a genetically derived nontoxic mutant of C. diphtheriae) (34) and by succinylation of the proteins prior to binding to the polysaccharide (27, 42, 43). Succinylation of a mutant Clostridium difficile toxin increased its solubility and its effectiveness for conjugates (43). Succinylation has been proposed for inactivating diphtheria and tetanus toxins and stabilizing the resultant toxoids (51). We evaluated the safety and immunogenicity of S. sonnei and S. flexneri type 2a conjugates in adults prepared with these two carrier proteins, native or treated with succinic anhydride. These agents were approved for investigation by the National Institutes of Health (OH98-CH-N009), Food and Drug Administration (BB IND 7443), Office for Protection against Research Risks (SPA SF-5900-09), and Ministry of Health, Israel.

Vaccination of chinchillas with nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) conjugates protected against otitis media. Correlations between the levels of conjugate-induced LOS antibodies (Abs) in sera and middle ear fluids (MEFs) and Ab-mediated biological functions and protection were examined. Following parenteral vaccination and middle ear challenge, all vaccinated animals, but none of the controls, had high titers of anti-LOS in their sera and MEFs. There was a correlation between the levels of anti-LOS IgG + M, IgG or IgA in the sera and in the MEFs (P0.001). An inverse correlation was found between the level of serum IgG + M and bacterial counts and between the levels of MEF Abs and bacterial counts at the early postchallenge stage (P0.05). Of the 39 vaccinated animals, 44% showed complete protection against otitis media, 46% (18/39) of their sera inhibited adherence of NTHi to human epithelial cells, 49% (19/39) demonstrated bactericidal activity and 49% (19/39) showed opsonophagocytic activity. In contrast, none of the controls (19) were protected, none of their sera inhibited bacterial adherence or had bactericidal activity and only 21% showed opsonophagocytosis. Our interpretation is that vaccine-induced LOS Abs transuded into the middle ear and conferred immunity to NTHi by binding to LOS of NTHi, inhibition of NTHi adherence to epithelial cells and complement-mediated bacteriolysis (or opsonophagocytosis).

Escherichia coli O157 is the major cause of diarrhea-associated hemolytic uremic syndrome (HUS). Strains causing HUS contain either Shiga toxin 1 (Stx1) or Stx2, or both. In adult volunteers, conjugate vaccines of detoxified lipopolysaccharide (LPS) elicited bactericidal antibodies to E. coli O157. Here, the detoxified LPS was conjugated with improved schemes to the nontoxic B subunit of Stx1. Mice injected with these bivalent conjugates elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx1.

The capsular polysaccharide of Salmonella typhi, Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with Pseudomonas aeruginosa recombinant exoprotein A ( r EPA) as the carrier, using either N -succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi- r EPA 1 ) or adipic acid dihydrazide (ADH; Vi- r EPA 2 ) as linkers. None of the recipients experienced a temperature of >38.5°C or significant local reactions. One injection of Vi- r EPA 2 into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi- r EPA 2 , Vi- r EPA 1 , and Vi were 169, 22.8, and 18.9 EU, respectively ( P = 0.0001 for Vi- r EPA 1 and Vi with respect to Vi- r EPA 2 ). At 26 weeks, the anti-Vi IgG levels for recipients of Vi- r EPA 2 , Vi- r EPA 1 , and Vi were 30.0, 10.8, and 13.4 EU, respectively ( P < 0.001 for Vi- r EPA 1 and Vi with respect to Vi- r EPA 2 ); all were higher than the preinjection levels ( P = 0.0001). Vi- r EPA 2 also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi- r EPA 2 elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi- r EPA 1 ( P = 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi- r EPA 2 and from 28.9 to 83.0 EU for Vi- r EPA 1 . At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi- r EPA 2 and 12.8 versus 0.33 for Vi- r EPA 1 ; P = 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi- r EPA 2 in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4; P = 0.0001). The safety and immunogenicity of the Vi- r EPA 2 conjugate warrant further investigation.

O-specific polysaccharide conjugates of shigellae were safe and immunogenic in young adults, and a Shigella sonnei conjugate conferred protection [1-3]. Shigellosis is primarily a disease of children; therefore, the safety and immunogenicity of S. sonnei and Shigella flexneri 2a conjugates were studied in 4- to 7-year-old children. Local and systemic reactions were minimal. The first injection of both conjugates elicited significant rises in geometric mean levels of serum IgG only to the homologous lipopolysaccharide (LPS) (S. sonnei, 0.32-8.25 ELISA units [EU]; S. flexneri 2a, 1.15-20.5 EU; P

Levels of IgG antibody to pertussis toxin (PT) and filamentous hemagglutinin (FHA) were measured in paired serum samples from 781 patients fulfilling at least one laboratory criterion for pertussis that was suggested by an ad hoc committee sponsored by the World Health Organization. The patients were participants or family members of participants in a double-blind efficacy trial of a monocomponent pertussis toxoid vaccine. Of 596 nonvaccinated children, 90% had significant (two-fold or more) rises in PT IgG and FHA IgG levels. Only 17 (32%) of 53 children previously vaccinated with three doses of pertussis toxoid had rises in PT IgG levels because they already had elevated PT IgG levels in their acute-phase serum samples. PT IgG and FHA IgG levels were significantly higher in acute-phase serum samples from 29 adults than in acute-phase serum samples from the nonvaccinated children. Nevertheless, significant rises in levels of PT IgG (79% of samples) and FHA IgG (90%) were demonstrated in adults. In conclusion, assay of PT IgG and FHA IgG in paired serum samples is highly sensitive for diagnosing pertussis in nonvaccinated individuals. Assay of PT IgG levels in paired sera is significantly less sensitive for diagnosis of pertussis for children vaccinated with pertussis toxoid.

l. Lenke RR, Levy HL. Maternal phenylketonuria and hyperphenylalaninemia: an international survey of the outcome of untreated and treated pregnancies. N Engl J Med 1980; 303:1202-8. 2. Drogari E, Smith 1, Beasley M, Lloyd JK. Timing of strict diet in relation to fetal damage in maternal phenylketonuria. Lancet 1987;2:927-30. 3. Bickel H. Neonatal mass screening for inborn errors of metabolism. In: Recent progress in perinatal medicine and prevention of congenital anomaly. IYC Commemorative Congress; Oct. 2t-22, 1979. Tokyo: Ministry of Health and Welfare, Government of Japan, Medical Information Service, Inc, 1979:260-9. 4. Levy HL, Waisbren SE. Effects of untreated maternal phenylketonuria and hyperphenylalaninemia on the fetus. N Engl J Med 1983;309:1269-74. 5. Kirkman HN, Hicks RE. More on untreated maternal hyperphenylala ninemia. N Engl J Med 1984;311:1125-6. 6. Pardridge WM. The safety of aspartame [Letter]. JAMA 1986; 256:2678. 7. Buist NRM, Tuerck J, Lis E, Penn R. Effects of untreated maternal phenylketonuria and hyperphenylalaninemia on the fetus. N Engl J Med 1984;3t 1:52-3. 8. Nie NH, Hull CH, Jenkins JG, Steinbrenner K, Bent DH. SPSS: Statistical Package for the Social Sciences. 2d Ed. New York: McGraw-Hill, 1975. 9. Koch R, Friedman EG, Wenz E, Jew K, Crowley C, Donnell G. Maternal phenylketonuria. J Inher Metab Dis 1986;9(suppl 2):159-68.

Conjugate vaccines were prepared by binding hydrazine-treated lipopolysaccharide (DeALPS) from Vibrio cholerae O1, serotype Inaba, to cholera toxin (CT) variants CT-1 and CT-2. Volunteers ( n = 75) were injected with either 25 μg of DeALPS, alone or as a conjugate, or the licensed cellular vaccine containing 4 × 10 9 organisms each of serotypes Inaba and Ogawa per ml. No serious adverse reactions were observed. DeALPS alone did not elicit serum LPS or vibriocidal antibodies in mice and only low levels of immunoglobulin M (IgM) anti-LPS in the volunteers. Recipients of the cellular vaccine had the highest IgM anti-LPS levels, but the difference was not statistically significant from that elicited by the conjugates. The conjugates elicited the highest levels of IgG anti-LPS (DeALPS-CT-2 > DeALPS-CT-1 > cellular vaccine). Both conjugates and the cellular vaccine elicited vibriocidal antibodies: after 8 months, recipients of cellular vaccine had the highest geometric mean titer (1,249), followed by DeALPS-CT-2 (588) and DeALPS-CT-1 (330). The correlation coefficient between IgG anti-LPS and 2-mercaptoethanol (2-ME)-resistant vibriocidal antibodies was 0.81 ( P = 0.0004). Convalescent sera from cholera patients had a mean vibriocidal titer of 2,525 that was removed by treatment with 2-ME. The vibriocidal activities of sera from all vaccine groups and from the patients were absorbed (>75%) by LPS but not by either CT-1 or CT-2. Conjugate-induced IgG vibriocidal antibodies persisted longer than those elicited by the whole-cell vaccine. Both conjugates, but not the cellular vaccine, elicited IgG anti-CT.

Objective. In a previously reported double blind placebo-controlled trial it was shown that vaccination with pertussis toxoid during infancy reduced the incidence of pertussis in the vaccinees. Parents and siblings of participants in the trial were followed for pertussis to determine whether vaccination provided indirect protection of close contacts in a nonvaccinating country with a high incidence of pertussis. Study design. A group of 3450 infants were randomized to vaccination with diphtheria, tetanus and pertussis toxoids (DTPtxd) or to diphtheria and tetanus toxoids (DT). Pertussis cases were actively sought and diagnosed by cultures and serology in vaccinees (previously reported) and in family members during 2 years after the third vaccination. Results. Pertussis as defined by the World Health Organization (paroxysmal cough of ≥21 days and certain laboratory criteria) was diagnosed in 11 parents of DTPtxd recipients and in 26 parents of DT recipients; indirect protection was 60% (95% confidence intervals, 16 to 82%). In nonvaccinated younger siblings of DTPtxd and DT recipients there were 10 and 18 cases of pertussis, respectively; indirect protection was 43% (95% confidence intervals, −31 to 76%). When all cases of pertussis with cough ≥7 days were included, the indirect protection was 44% (95% confidence intervals, 7 to 67%) in parents and 56% (95% confidence intervals, 9 to 81%) in younger siblings. Conclusion. Vaccination of children with pertussis toxoid reduces spread of pertussis to close contacts, which suggests that mass vaccination with pertussis toxoid would induce herd immunity.

Detoxified-lipooligosaccharide (dLOS)-protein conjugates from nontypeable Haemophilus influenzae (NTHi) elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (X.-X. Gu, C.-M. Tsai, T. Ueyama, S. J. Barenkamp, J. B. Robbins, and D. J. Lim, Infect. Immun. 64:4047-4053, 1996). In this study, we evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-conjugated tetanus toxoid, dLOS-conjugated high-molecular-weight proteins from NTHi, or saline (control) in Freund's adjuvant and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS antibody, and 49% (19 of 39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all 19 controls and 56% (22 of 39) of the vaccinated animals during a period of 21 days (P < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (P < 0.01). The incidences of infection in the unchallenged ear or inner ear were 26 or 28% in the vaccine groups and 53 or 58% in the controls (P < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicate that active immunization with LOS-based conjugates reduces the incidence of NTHi-induced otitis media.

Despite the availability of a safe, effective polysaccharide vaccine, group A meningococcal meningitis epidemics persist in sub-Saharan Africa. In October 1996, there were almost 150,000 reported cases and 15,000 deaths, the majority of which involved children. At 3 months of age, induction of protective group A meningococcal antibody levels requires 2 injections at least 1 month apart. Reinjection of 5-year-old children increases group A antibodies to long-term protective levels. During meningitis epidemics in Nigeria, Mali, and Rwanda, fatality was significantly reduced in areas where scarce vaccine was administered selectively. Although effective on an individual basis, selective vaccination is unable to control meningitis epidemics. In Chad, mass vaccination of the entire population (excluding infants under 12 months) eliminated the disease. Successful mass vaccination against group A meningococcal epidemics also has been reported in Saudi Arabia, China, and refugee camps in Africa. Although cost is cited as an obstacle to routine mass vaccination to prevent meningococcal meningitis in South Africa, prevention is the least expensive approach to disease control. It is recommended that the entire population of Africa's meningitis belt receive group A meningococcal vaccine in accordance with the recommended age schedule in a mass vaccination program.

The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals > or = 5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-alpha(1-->4)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 microg, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5- to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast beta-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.

Summary Background The aim of this double-blind randomised vaccine-controlled trial was to assess the efficacy of a conjugate vaccine composed of Shigella sonnei O-specific polysaccharide bound to Pseudomonas aeruginosa recombinant exoprotein A ( S sonnei -r EPA) and of an oral, live-attenuated Escherichia coli/S flexneri 2a ( EcSf 2a-2) hybrid vaccine among military recruits in Israel at high risk of exposure to Shigella spp. We report here our preliminary findings on the efficacy of S sonnei -rEPA; we have not documented sufficient cases to assess the efficacy of EcSf 2a-2. Methods Between April, 1993, and August, 1994, male Israeli military recruits aged 18–22 years were asked to take part in our study. We enrolled 1446 soldiers from seven separate field sites (groups A–G). Soldiers were randomly allocated one injection of S sonnei -rEPA and four doses of oral placebo (n=576), four oral doses of EcSf 2a-2 and one injection of saline placebo (n=580), or one injection of meningococcal tetravalent control vaccine and four doses of oral placebo (n=290). Because there were no cases of S flexneri 2a, the EcSf 2a-2 and meningococcal vaccines were the control group. We defined S sonnei shigellosis as diarrhoea with a positive faecal culture for S sonnei. Each group of soldiers was followed up for 2·5–7·0 months. The primary endpoint was protective efficacy of S sonnei -rEPA against S sonnei shigellosis. Findings Cases of culture-proven S sonnei shigellosis occurred in four groups of soldiers (groups A–D), which comprised 787 volunteers (312 received S sonnei -rEPA, 316 received EcSf 2a-2, and 159 received meningococcal control vaccine). In groups A–C, cases of shigellosis occurred 70–155 days after vaccination, whereas in group D cases occurred after 1–17 days. In groups A–C, the attack rate of shigellosis was 2·2% in recipients of S sonnei -rEPA compared with 8·6% in controls (protective efficacy 74% [95% CI 28–100], p=0·006). S sonnei -rEPA also showed significant protection against shigellosis in group D (43% [4–82], p=0·039). Prevaccination and postvaccination ELISA measurements of antibody to S sonnei lipopolysaccharide among recipients of S sonnei -rEPA showed that the vaccinees who developed S sonnei shigellosis had significantly lower serum IgG and IgA responses to the homologous lipopolysaccharide than those who did not (p=≤0·05). Interpretation One injection of S sonnei -rEPA confers type-specific protection against S sonnei shigellosis. The high antibody concentration induced by the conjugate vaccine in volunteers who did not develop shigellosis suggests that there is an association between serum antibody titre and protection.