Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri†3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin.

BACKGROUND: Rosetting, namely the capacity of the Plasmodium falciparum-infected red blood cells to bind uninfected RBCs, is commonly observed in African children with severe malaria. Rosetting results from specific interactions between a subset of variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins encoded by var genes, serum components and RBC receptors. Rosette formation is a redundant phenotype, as there exists more than one var gene encoding a rosette-mediating PfEMP1 in each genome and hence a diverse array of underlying interactions. Moreover, field diversity creates a large panel of rosetting-associated serotypes and studies with human immune sera indicate that surface-reacting antibodies are essentially variant-specific. To gain better insight into the interactions involved in rosetting and map surface epitopes, a panel of monoclonal antibodies (mAbs) was investigated. METHODS: Monoclonal antibodies were isolated from mice immunized with PfEMP1-VarO recombinant domains. They were characterized using ELISA and reactivity with the native PfEMP1-VarO adhesin on immunoblots of reduced and unreduced extracts, as well as SDS-extracts of Palo Alto 89F5 VarO schizonts. Functionality was assessed using inhibition of Palo Alto 89F5 VarO rosette formation and disruption of Palo Alto 89F5 VarO rosettes. Competition ELISAs were performed with biotinylated antibodies against DBL1 to identify reactivity groups. Specificity of mAbs reacting with the DBL1 adhesion domain was explored using recombinant proteins carrying mutations abolishing RBC binding or binding to heparin, a potent inhibitor of rosette formation. RESULTS: Domain-specific, surface-reacting mAbs were obtained for four individual domains (DBL1, CIDR1, DBL2, DBL4). Monoclonal antibodies reacting with DBL1 potently inhibited the formation of rosettes and disrupted Palo Alto 89F5 VarO rosettes. Most surface-reactive mAbs and all mAbs interfering with rosetting reacted on parasite immunoblots with disulfide bond-dependent PfEMP1 epitopes. Based on competition ELISA and binding to mutant DBL1 domains, two distinct binding sites for rosette-disrupting mAbs were identified in close proximity to the RBC-binding site. CONCLUSIONS: Rosette-inhibitory antibodies bind to conformation-dependent epitopes located close to the RBC-binding site and distant from the heparin-binding site. These results provide novel clues for a rational intervention strategy that targets rosetting.

Non-stoichiometric O-acetylation of Shigella flexneri 2a O-specific polysaccharide: synthesis and antigenicity.

Synthetic functional mimics of the O-antigen from Shigella flexneri 2a are seen as promising vaccine components against endemic shigellosis. Herein, the influence of the polysaccharide non-stoichiometric di-O-acetylation on antigenicity is addressed for the first time. Three decasaccharides, representing relevant internal mono- and di-O-acetylation profiles of the O-antigen, were synthesized from a pivotal protected decasaccharide designed to tailor late stage site-selective O-acetylation. The latter was obtained via a convergent route involving the imidate glycosylation chemistry. Binding studies to five protective mIgGs showed that none of the acetates adds significantly to broad antibody recognition. Yet, one of the five antibodies had a unique pattern of binding. With IC50 in the micromolar to submicromolar range mIgG F22-4 exemplifies a remarkable tight binding antibody against diversely O-acetylated and non-O-acetylated fragments of a neutral polysaccharide of medical importance.

A five-year field assessment of rapid diagnostic tests for meningococcal meningitis in Niger by using the combination of conventional and real-time PCR assays as a gold standard.

BACKGROUND: Meningococcocal meningitis represents an important cause of mortality and morbidity in sub-Saharan countries. Confirmatory bacteriological or molecular diagnosis is essential for patient management/treatment and meningitis surveillance, but many laboratory tests are expensive and rarely available for low-income countries. A rapid diagnostic test (RDT) represents a valuable alternative to improve case management and surveillance. METHODS: A dipstick RDT developed in early 2000s that detects Neisseria meningitidis serogroups A, C, W and Y but for which a new conjugated antibody (L4-8) for the detection of serogroup A replaced the original K15-2 was assessed in the field by trained staff from health centres and district hospitals in Niger. The results were compared to those obtained in the reference laboratory and the sensitivity and specificity of RDTs were determined using conventional and real-time PCR assays as a gold standard. RESULTS: RDT results from field staff and the reference laboratory obtained for 2095 cerebrospinal fluid (CSF) specimens presented a strong concordance of 94% with Cohen's κ coefficient of 0.88. The observed concordance between RDTs operated by staff from the reference laboratory vs combination of conventional and real-time PCR assays was 89% with Cohen's κ coefficient of 0.76 indicating very good agreement. The theoretical overall sensitivity for RDT was 91.5% and the specificity 84.6%. CONCLUSIONS: RDT has proven to be relatively sensitive and specific for the detection of meningococcal serogroups A/C/Y/W. We confirmed that these RDTs can be reliably operated by trained but non-specialised staff in basic health facilities.

Laboratory evaluation of a rapid diagnostic test for Neisseria meningitidis serogroup A.

BACKGROUND: Epidemics of meningococcal meningitis occur periodically in the African 'meningitis belt' and are mainly, but not only, due to serogroup A. METHODS: We tested a dipstick as a rapid detection test (RDT) to detect serogroup A using 401 cerebrospinal fluid (CSF) samples. RESULTS: The detection limits were 10(5) CFU/ml with sensitivity and specificity for detecting serogroup A in CSF samples of 88% and 99%, respectively. CONCLUSIONS: The new RDT can be used in field surveillance of meningococcal meningitis to help characterize meningitis cases particularly after introduction of the conjugate vaccine against serogroup A.

Enhanced basophil reactivities during severe malaria and their relationship with the Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein.

Recent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derived Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruited P. falciparum-infected patients in Senegal with mild malaria (MM; n = 19) or severe malaria (SM; n = 9) symptoms and healthy controls (HC; n = 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measured ex vivo using fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.

Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis.

BACKGROUND: Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. METHODS: S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. RESULTS: We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. CONCLUSIONS: This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance.

Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments. Additionally, these complementary pieces of information give evidence to the ability of the studied mAb to recognize internal as well as terminal epitopes of its cognate polysaccharide antigen. Hence, we show that an appropriate combination of computational and experimental methods provides a basis to explore carbohydrate functional mimicry and receptor binding. The strategy may facilitate the design of either ligands or carbohydrate recognition domains, according to needed improvements of the natural carbohydrate:receptor properties.

An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype.

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1alpha(1) (DBL1alpha(1)) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1alpha(1) recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1alpha(1)-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1alpha(1) domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

Biological diagnosis of meningococcal meningitis in the African meningitis belt: current epidemic strategy and new perspectives.

Laboratory diagnosis is an essential component in surveillance of meningococcal epidemics, as it can inform decision-makers of the Neisseria meningitidis serogroup(s) involved and the most appropriate vaccine to be selected for mass vaccination. However, countries most affected face real limitations in laboratory diagnostics, due to lack of resources. We describe current diagnostic tools and examine their cost-effectiveness for use in an epidemic context. The conclusion is that current WHO recommendations to use only the latex agglutination assay (Pastorex) at epidemic onset is cost-effective, but recently developed rapid diagnostic tests for the major epidemic-causing meningococcal serogroups may prove a breakthrough for the future.

New rapid diagnostic tests for Neisseria meningitidis serogroups A, W135, C, and Y.

BACKGROUND: Outbreaks of meningococcal meningitis (meningitis caused by Neisseria meningitidis) are a major public health concern in the African "meningitis belt," which includes 21 countries from Senegal to Ethiopia. Of the several species that can cause meningitis, N. meningitidis is the most important cause of epidemics in this region. In choosing the appropriate vaccine, accurate N. meningitidis serogroup determination is key. To this end, we developed and evaluated two duplex rapid diagnostic tests (RDTs) for detecting N. meningitidis polysaccharide (PS) antigens of several important serogroups. METHODS AND FINDINGS: Mouse monoclonal IgG antibodies against N. meningitidis PS A, W135/Y, Y, and C were used to develop two immunochromatography duplex RDTs, RDT1 (to detect serogroups A and W135/Y) and RDT2 (to detect serogroups C and Y). Standards for Reporting of Diagnostic Accuracy criteria were used to determine diagnostic accuracy of RDTs on reference strains and cerebrospinal fluid (CSF) samples using culture and PCR, respectively, as reference tests. The cutoffs were 10(5) cfu/ml for reference strains and 1 ng/ml for PS. Sensitivities and specificities were 100% for reference strains, and 93.8%-100% for CSF serogroups A, W135, and Y in CSF. For CSF serogroup A, the positive and negative likelihood ratios (+/- 95% confidence intervals [CIs]) were 31.867 (16.1-63.1) and 0.065 (0.04-0.104), respectively, and the diagnostic odds ratio (+/- 95% CI) was 492.9 (207.2-1,172.5). For CSF serogroups W135 and Y, the positive likelihood ratio was 159.6 (51.7-493.3) Both RDTs were equally reliable at 25 degrees C and 45 degrees C. CONCLUSIONS: These RDTs are important new bedside diagnostic tools for surveillance of meningococcus serogroups A and W135, the two serogroups that are responsible for major epidemics in Africa.

Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population.

BACKGROUND: Early detection of cholera outbreaks is crucial for the implementation of the most appropriate control strategies. METHODS: The performance of an immunochromatographic dipstick test (Institute Pasteur, Paris, France) specific for Vibrio cholerae O1 was evaluated in a prospective study in Beira, Mozambique, during the 2004 cholera season (January-May). Fecal specimens were collected from 391 patients with acute watery nonbloody diarrhea and tested by dipstick and conventional culture. RESULTS: The overall sensitivity and specificity of the rapid test compared to culture were 95% (95% confidence interval [CI]: 91%-99%) and 89% (95% CI: 86%-93%), respectively. After stratification by type of sample (rectal swab/bulk stool) and severity of diarrhea, the sensitivity ranged between 85% and 98% and specificity between 77% and 97%. CONCLUSION: This one-step dipstick test performed well in the diagnosis of V. cholerae O1 in a setting with seasonal outbreaks where rapid tests are most urgently needed.

Evaluation of three rapid diagnostic tests for cholera: does the skill level of the technician matter?

OBJECTIVE: To evaluate SMART, Medicos Dip Stick and an Institut Pasteur (IP) cholera dipstick tests for accuracy and ease of use. METHOD: Every 50th patient presenting with diarrhoea at ICDDR,B between 1 April 2003 and 30 November 2003 was enrolled. The rapid diagnostic tests were performed by field and laboratory technicians, and sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values calculated. RESULTS: We isolated Vibrio cholerae O1 from 116 (38%) of 304 patients. The Se, Sp, PPV and NPV of the SMART test were 58%, 95%, 84% and 84% for field technicians, and 83%, 88%, 83% and 88% for laboratory technicians. The Se, Sp, PPV and NPV of the IP dipstick test were 93%, 67%, 63% and 94% for field technicians, and 94%, 76%, 70% and 95% for laboratory technicians. The Se, Sp, PPV and NPV of the Medicos test were 84%, 79%, 71% and 90% for field technicians, and 88%, 80%, 72% and 92% for laboratory technicians. A high proportion of indeterminates (30%) hampered the performance of the SMART test. The IP dipstick had the highest Se, irrespective of technician skill level. CONCLUSION: The IP dipstick is the most appropriate rapid diagnostic assay for the detection of V. cholerae O1 in locations where the skill level of personnel may be low, such as remote areas or refugee camp settings. High cost may limit the utility of any diagnostic test in the developing world.

Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine.

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.

Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.

The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues.

Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs.

We evaluated the recently developed dipsticks for the rapid detection of Vibrio cholerae serotypes O1 and O139 from rectal swabs of hospitalized diarrheal patients after enrichment for 4 h in alkaline peptone water. The sensitivity and specificity of the dipsticks were above 92 and 91%, respectively. The dipsticks represent the first rapid test which has been successfully used to diagnose cholera from rectal swabs, and this would immensely improve surveillance for cholera, especially in remote settings.

Development and testing of a rapid diagnostic test for bubonic and pneumonic plague.

BACKGROUND: Plague is often fatal without prompt and appropriate treatment. It affects mainly poor and remote populations. Late diagnosis is one of the major causes of human death and spread of the disease, since it limits the effectiveness of control measures. We aimed to develop and assess a rapid diagnostic test (RDT) for plague. METHODS: We developed a test that used monoclonal antibodies to the F1 antigen of Yersinia pestis. Sensitivity and specificity were assessed with a range of bacterial cultures and clinical samples, and compared with findings from available ELISA and bacteriological tests for plague. Samples from patients thought to have plague were tested with the RDT in the laboratory and by health workers in 26 pilot sites in Madagascar. FINDINGS: The RDT detected concentrations of F1 antigen as low as 0.5 ng/mL in up to 15 min, and had a shelf life of 21 days at 60 degrees C. Its sensitivity and specificity were both 100%. RDT detected 41.6% and 31% more positive clinical specimens than did bacteriological methods and ELISA, respectively. The agreement rate between tests done at remote centres and in the laboratory was 89.8%. With the combination of bacteriological methods and F1 ELISA as reference standard, the positive and negative predictive values of the RDT were 90.6% and 86.7%, respectively. INTERPRETATION: Our RDT is a specific, sensitive, and reliable test that can easily be done by health workers at the patient's bedside, for the rapid diagnosis of pneumonic and bubonic plague. This test will be of key importance for the control of plague in endemic countries.

The binding of synthetic analogs of the upstream, terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS).

The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy. The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding. Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V. cholerae O:1 serotype Inaba was redefined. We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies. The results obtained allow further rationalization of the structural basis for the binding of V. cholerae O:1 antigens to their homologous antibodies.