WO2018130855A1 - Biomarqueurs typhoïdes - Google Patents

Biomarqueurs typhoïdes Download PDF

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Publication number
WO2018130855A1
WO2018130855A1 PCT/GB2018/050106 GB2018050106W WO2018130855A1 WO 2018130855 A1 WO2018130855 A1 WO 2018130855A1 GB 2018050106 W GB2018050106 W GB 2018050106W WO 2018130855 A1 WO2018130855 A1 WO 2018130855A1
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sty
typhoid
antibodies
seq
sample
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Stephen Baker
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Oxford University Innovation Limited
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel biomarker combinations for use in the diagnosis of typhoid fever.
  • novel biomarker combinations for use in the diagnosis of typhoid fever.
  • Enteric (typhoid) fever is a systemic infection caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S . Paratyphi A).
  • Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A S . Paratyphi A.
  • S . Paratyphi A Salmonella enterica serovars Typhi
  • S . Paratyphi A S typhoid fever
  • S typhoid fever remains the most commonly reported etiological agent of enteric fever in Asia and Africa.
  • Typhoid fever occurs only in humans, making it a disease that can technically be eradicated. Indeed, typhoid fever has all but been eliminated from several countries in Southeast Asia where it was the most common cause of hospitalised febrile disease 20-30 years ago. Elimination in these areas is generally attributed to extensive improvements in sanitation rather than en masse immunization programmes. Limited data regarding the long-term impact of mass immunization for typhoid fever and the performance of licensed vaccines has hindered immunization as a sustainable typhoid control and elimination strategy. The future consideration of rational control measures for typhoid fever (including the introduction of new conjugate vaccines) will rely on accurately assessing disease burden, which requires a reliable diagnostic approach.
  • a further approach for typhoid diagnostics is the use of serological assays, i.e. the detection of antibody against the infecting pathogen.
  • serological assays i.e. the detection of antibody against the infecting pathogen.
  • a range of serological RDTs to identify patients with typhoid fever are available, these tests generally detect antibody against Salmonella flagellin (H-antigen) or Lipopolysaccharide (LPS; O-antigen).
  • H-antigen Salmonella flagellin
  • LPS Lipopolysaccharide
  • these antigens are highly cross-reactive with other members of the Enterobacteriaceae, giving less than desirable operational levels of sensitivity and specificity for typhoid fever diagnosis.
  • An aim of the present invention is to provide alternative and more specific markers for use in the detection of typhoid fever.
  • the present invention provides a method of determining the typhoid status of a subject comprising:
  • STY0452 SEQ ID NO: 1
  • STY0796 SEQ ID NO: 2
  • STY 1086 SEQ ID NO: 3
  • STY 1372 SEQ ID NO: 4
  • STY 1612 SEQ ID NO: 5
  • STY4539 SEQ ID NO: 6
  • STY 1522 SEQ ID NO: 7
  • STY 1703 SEQ ID NO: 8
  • STY 1767 SEQ ID NO: 9
  • STY 1886 SEQ ID NO : 10
  • STY3208 SEQ ID NO : 1 1
  • STY4910 SEQ ID NO: 12
  • the Vi polysaccharide of Salmonella Typhi is a linear homopolymer of poly- alpha( l ⁇ 4)GalNAcp variably O acetylated at the C-3 position.
  • the method of the invention is for determining whether a subject has typhoid fever.
  • the method of the invention is for determining whether a subject has an active S . Typhi infection.
  • step (b) the presence of antibodies to the expression product of at least one of STY4539 (PilL), STY1886 (CdtB) and STY 1703 are determined.
  • the presence of antibodies to the expression product of STY4539 (PilL) and STY1886 (CdtB) are determined in (b).
  • the presence of antibodies to the expression product of STY 1703 and STY 1886 (CdtB) are determined in (b).
  • step (b) the antibody titre to a specific antigen may be determined, rather than just the absence or presence of antibodies directed to a specific antigen.
  • step (d) the levels observed in (b) and (c) may be compared to a control.
  • the control may be the levels of antibodies observed in the local population who do not have typhoid disease.
  • the method may be used to detect IgM antibodies.
  • the level of antibody may be determined by any appropriate method, including immunoassay, spectrometry, western blot, ELISA, immunoprecipitation, isoelectric focusing, SDS-PAGE, radioimmunoassay (RIA), fluoroimmunoassay, surface enhanced Raman spectroscopy (SERS), or combinations thereof.
  • immunoassay spectrometry, western blot, ELISA, immunoprecipitation, isoelectric focusing, SDS-PAGE, radioimmunoassay (RIA), fluoroimmunoassay, surface enhanced Raman spectroscopy (SERS), or combinations thereof.
  • the presence, and optionally the level, of the antibody biomarkers in a sample may be determined by using epitopes from the expression products of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or an antigenic part therof.
  • the expression products of the genes are preferably proteins.
  • the sample may be a sample of blood, serum, plasma, sputum or saliva obtained from a subject.
  • the method of the invention does not include the step of obtaining the sample .
  • the sample may be obtained from a mammal, the mammal may be human.
  • the method of the invention is carried out in vitro.
  • a sensitivity of at least 80%, preferably at least 85%, preferably at least 90%, for the detection of typhoid fever can be achieved.
  • Sensitivity measures the proportion of positives that are correctly identified as such (e.g., the percentage of sick people who are correctly identified as having typhoid fever), specificity measures the proportion of negatives that are correctly identified as such (e.g., the percentage of healthy people who are correctly identified as not having typhoid fever).
  • One advantage of the method of the invention is that it is much more accurate than current methods to detect typhoid fever. This means that patients can be given the optimal treatment and that the unnecessary use of antimicrobials can be reduced.
  • the method of the invention also has the advantage that areas where typhoid is occurring can be identified and targeted immunisation programmes can be introduced.
  • the method also allow the burden of the disease to more accurately assessed.
  • the method of the invention may be used to triage patients so that only those with typhoid fever are treated for typhoid fever. This will reduce the unnecessary administration of antibiotics to patients who do not have typhoid fever.
  • the method of the invention may also be used in combination with markers for other diseases, for example, markers for dengue fever and/or malaria and/or for any other diseases that present in a manner similar to typhoid fever.
  • the invention provides a biomarker panel for determining the typhoid status of a subject, wherein the panel comprises antibodies to the expression product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide.
  • the invention provides a method for detecting typhoid in an in vitro sample obtained from a subject, wherein the sample is contacted with a solid-state device onto which has been immobilised probes derived from the expression products of one or more of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide.
  • the probes may be proteins or peptides derived from the expression product of STY4539 (PilL) and/or STY1886 (CdtB) and/or STY 1703, preferably from STY4539 (PilL) and/or STY 1886 (CdtB).
  • the solid-state device may comprise a substrate having a probe or multiple different probes immobilised upon it that bind specifically to the biomarkers being screened for.
  • the probe may be a protein or a polypeptide, or polysaccharide.
  • the device may also comprise probes specific for other diseases, for example, dengue fever and/or malaria.
  • the substrate may be any surface able to support one or more probes. It may be polymer based, metallic, ceramic or any other suitable material.
  • the invention provides a method of detecting antibodies to the expression product of one or more genes selected from the group consisting of
  • the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and
  • the invention provides a method of diagnosing typhoid fever in a patient, said method comprising
  • the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and
  • the invention provides a method of diagnosing and treating typhoid fever in a subjecr, said method comprising
  • the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and
  • an effective amount of an antibiotic to the subject.
  • an antibiotic preferably fluoroquinolone and/or azithromycin.
  • the biomarker concentration values, or antibody titres, determined in the method of the invention are inputted into a statistical methodology to produce an output value that correlates with the chances that the patient has typhoid fever.
  • the statistical method considers the expected levels of the biomarkers that would be observed in a subject without typhoid fever. This may be based on levels seen in a control local population of subjects that do not have typhoid fever as determined by other means.
  • the statistical methodology used is logistic regression, decision trees, support vector machines, neural networks, random forest or another machine-learning algorithm.
  • the performance of the results of the applied statistical methods used in accordance with the present invention can be best described by their receiver operating characteristics (ROC).
  • ROC receiver operating characteristics
  • the ROC curve addresses both the sensitivity (the number of true positives) and the specificity (the number of true negatives) of the test. Therefore, sensitivity and specificity values for a given combination of biomarkers are an indication of the performance of the test.
  • a biomarker combination has a sensitivity and specificity value of 85 %, this means that out of 100 patients, 85 will be correctly identified/diagnosed from the determination of the presence of the particular combination of biomarkers as positive for the disease, while out of 100 patients who do not have the disease 85 will accurately test negative for the disease .
  • a suitable statistical classification model such as logistic regression, can be derived for a combination of biomarkers.
  • the logistic regression equation can be extended to include other (clinical) variables such as age and gender of the patient.
  • the ROC curve can be used to access the performance of the discrimination between patients and controls by the logistic regression model. Therefore, the logistic regression equation can be used apart or combined with other clinical characteristics to aid clinical decision making.
  • AUC area under the curve
  • the two different conditions can be whether a patient has or does not have typhoid fever.
  • the step of comparing the antibody levels in a sample with a control can comprise querying a database of reference levels of the test biomarkers from a plurality of reference samples, wherein the database of known antibody levels comprises the antibody levels of at least the test biomarkers for samples of bodily fluids taken from subjects diagnosed with typhoid disease and/or with samples from subjects who do not have typhoid.
  • the database comprises samples from at least 10 subjects with typhoid and 10 without.
  • the data base is queried by undertaking statistical analysis, such as a multivariate statistical analysis, preferably a least squares fit analysis such as a partial least squares regression analysis and more preferably a partial least squares discriminant analysis, of the known reference antibody levels against the antibody levels in the sample from the subject being tested/studied.
  • statistical analysis such as a multivariate statistical analysis, preferably a least squares fit analysis such as a partial least squares regression analysis and more preferably a partial least squares discriminant analysis, of the known reference antibody levels against the antibody levels in the sample from the subject being tested/studied.
  • At least one reference group antibody profile may be obtained by: analyzing reference samples obtained from patients with a diagnosis of typhoid or patients who do not have typhoid, measuring the reference samples for the reference antibody levels of at least the test biomarkers; undertaking statistical analysis of the reference antibody levels of each reference sample relative to the reference antibody levels of the ensemble of reference samples to determine a relative antibody profile for each reference sample; and generating a reference group antibody profile by mapping the relative antibody profile of each reference sample to the known typhoid status of each reference sample.
  • the step of calculating a statistical score may further comprise : undertaking statistical analysis of the antibody levels of the test biomarkers in the test sample relative to the antibody levels of the ensemble of reference samples to determine a relative test antibody profile for the test sample; and determining the statistical fit between the relative test antibody profile and the relative antibody profile and assigning a statistical score based on the statistical fit.
  • each newly obtained series of antibody levels of the test biomarkers is compared to the reference antibody levels for the test biomarkers and mapped onto the statistical distribution obtained between the reference antibody levels and profiles and the diagnosis associated with each reference antibody level and profile .
  • This allows a statistical score to be attributed to the obtained test sample relative to the reference samples or diagnoses used to generate the reference antibody levels and profiles. For example, a higher score may represent an increased likelihood that the test sample is from a subject suffering from typhoid.
  • the score may be a value that indicates how closely the test sample conforms to the antibody profile of the determined diagnosis.
  • the statistical analysis may use partial least squares fit techniques and optionally or preferably may use partial least squares discriminant analysis.
  • the statistical analysis may be a multivariate statistical analysis.
  • the statistical analysis may interrogate the internal correlation structure and relative antibody levels between the at least three biomarkers
  • other statistical techniques may be used relevant to the regression analysis performed. It can be appreciated that different statistical techniques may provide differing confidence intervals. The choice of statistical technique is typically dependent upon the suitability of the data.
  • a system for calculating the probability that a subject has typhoid comprising: a test sample of bodily fluid obtained from a subject; a panel of test biomarkers; a processor for undertaking statistical analysis on the antibody levels of the biomarkers from the panel; a database containing one or more reference group expression profiles; and an output device for signalling the results of the statistical analysis, wherein the processor determines a statistical score based on a comparison between the antibody levels of the panel of test biomarkers in the test sample and the reference group antibody profiles representing the probability that the antibody levels of the test biomarkers in the test sample diagnose typhoid.
  • the system may be used to assist in the diagnosis, prediction or monitoring of typhoid and may also form part of a test available to medical practitioners to assist diagnosis and monitor disease progression.
  • the method of the invention may be used to monitor the progression of typhoid fever in a subject or to monitor the response of a subject to treatment for typhoid fever.
  • the invention may provide a kit for determining the typhoid status of subject, wherein the kit comprises the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or an antigenic fragment of the Vi polysaccharide.
  • the peptides and polysaccharide or fragments thereof may be on a chip for high throughput screening.
  • the kit could comprise a multi-well plate or microfluidic card or multi-plex chip prepared with reagents to capture and quantify the markers constituting the biomarker panel or fingerprint, as well as a database containing disease reference profiles and a computer module facilitating comparison of the test results with the reference panel using appropriate statistics.
  • Equipment needed to read the plate or microfluidic card or chip would be standard high throughput laboratory equipment such as Luminex or Mesoscale Discovery or quantitative PCR or microarray platforms.
  • the kit may comprise instructions for suitable operational parameters in the form of a label or separate insert. The instructions may inform a consumer about how to collect the sample .
  • the skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention. Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying figures.
  • Figure 1 - depicts Table 1 which includes details of the plasmid constructs of Salmonella Typhi antigens generated and used in this invention.
  • Figure 2 - depicts Table 2 which includes details of the Salmonella Typhi antigens expressed in this study for serological testing.
  • Figure 5 - illustrates the correlation of IgM measurements between Salmonella Typhi antigens.
  • the data presented is a representative selection of the data obtained which shows a correlation in IgM measurements in human plasma for the antigens encoded by STY4539, STY 1703, STY 1886 and the Vi antigen.
  • the histograms show the distribution of IgM levels by optical density to the highlighted S. Typhi antigen.
  • the scatterplot above the histogram, plots IgM measurements between the two antigens on a right angle to the scatterplot and describes the correlation between antibody responses to two selected antigens.
  • the numerals below the histogram depict the Spearman correlation coefficient (rho) values of the mirrored scatterplot.
  • Figure 6 - illustrates IgM responses against Salmonella Typhi antigens in a Bangladeshi cohort of febrile patients and controls.
  • the boxplots show IgM measurements (optical density) in plasma in afebrile controls (light grey), febrile patients with an infection other than typhoid fever (medium grey) and typhoid patients (dark grey). Dark horizontal lines represent the mean, with the box representing the 25th and 75th percentiles, whiskers represent the 5th and 95th percentiles, and outliers are represented by dots.
  • Figure 6 A shows boxplots of antibody responses against (in order) STY0452, STY0769,
  • FIG. 7 - shows the results of experiments assessing the sensitivity and specificity of IgM against Salmonella Typhi antigens for the diagnosis of typhoid fever.
  • Receiver operating characteristic (ROC) curves summarise the antibody responses against antigen combinations for the diagnosis of typhoid fever.
  • the x-axis displays the false positivity rate (Specificity) and the y-axis displays true positive rate (Sensitivity).
  • the performance of two, three and four antigens are shown by the dotted, light grey and dark grey lines, respectively.
  • Figure 8 - demonstrates that febrile patients with an IgM profile indicative of typhoid fever can be detected using the invention.
  • the hyperlane plots predict the number of undiagnosed febrile patients that have an IgM measurement indicative of typhoid fever.
  • the black circles represent the negative controls, which includes healthy controls and patients other with infections.
  • the white squares represent typhoid cases confirmed by blood culture or PCR.
  • the white triangles are febrile patients with an IgM profile indicative of not having typhoid fever, whilst the grey triangles are febrile patients defined as having a typhoid infection using pre-defined hyperlane IgM profile .
  • Figure 9 - shows the correlation of IgM measurements between Salmonella Typhi antigens.
  • the plots show a correlation in IgM measurements in human plasma for the antigens encoded by STY0452, STY0769, STY 1086, STY 1372,
  • CMCH Chittagong Medical College Hospital
  • gold standards for typhoid fever diagnosis are blood culture and PCR amplification from blood using a previously described method - Mga et al (2010) BMC Infect Dis 10: 125.
  • Blood (5- 12 mL for adults and 1- 12 mL) was cultured using Bact/Alert-FA and PF blood culture bottles, bottles were incubated in the Bact/Alert automated system (Biomerieux, Marcy l'Etoile, France) for five days. The patient demographics and diagnostic testing results for this study are reported elsewhere Maude et al. (2015) Trop Med Int Health 20: 1376-84.
  • plasma samples from 40 healthy adult control subjects 32 cases of confirmed typhoid fever ( 16 cases confirmed by blood culture, 13 cases confirmed by PCR and three cases confirmed by both blood culture and PCR), 17 cases from patients with confirmed febrile diseases other than typhoid fever and 243 febrile patients with undiagnosed conditions (332 patient samples in total) were subjected to serological assays using the S. Typhi antigens.
  • ⁇ 2 S. Typhi antigens were selected that gave a differential serodiagnostic signal using protein microarray screening for further expression and purification (Table 1 in Figure 1).
  • the coding sequences of the selected genes excluding trans-membrane domains and other proteomic features likely to hinder protein solubility and stop codons, were cloned into the 5 ' Ncol and 3 ' Noil restriction sites of pET28b vector (Novagen, United Kingdom) for further His-Tag purification.
  • E. coli DH5a were transformed with the plasmid constructs for stable storage and E. coli BL21 -DE3 PLYSS (Promega, WI, USA) were used for expression and purification.
  • the predicted protein sizes ranged from 5.7 KDal to 52.6 KDal in size (Table li n Figure 1).
  • E. coli BL21 -DE3 PLYSS strains harboring unique plasmid constructs (pEK90-pEK109) containing the genes of interest were inoculated into Luria-Bertani (LB) broth containing l OO mg/L kanamycin (Sigma, MO, USA), and incubated at 37°C overnight with gentle circular agitation (40-50rpm). Overnight cultures were diluted ( 1 : 100) into LB broth and incubated at 37°C with agitation until an optical density (OD 60 o) of 0.5 was reached.
  • LB Luria-Bertani
  • kanamycin Sigma, MO, USA
  • IPTG isopropyl- -D-thiogalactoside
  • soluble proteins bacterial pellets were resuspended in 50 mM phosphate buffer (pH 8) containing 300 mM NaCl and 10 mM imidazole . The suspension was gently agitated at ambient temperature for 30 minutes prior to sonication (3 cycles of 10 seconds for 2 minutes with 10 seconds in between cycles). Cell debris and the membrane fragment were pelleted by centrifugation at 16,000 x g at 4°C for 30 minutes and discarded. Supernatants were filtered through a 0.45 ⁇ membrane before being rocked at 4°C with nickel coated agarose beads (Ni-NTA, Invitrogen) for two hours.
  • Ni-NTA nickel coated agarose beads
  • Protein bound Ni-NTA beads were loaded into gravity flow columns (Qiagen, Hilden, Germany) and washed with 20mM imidazole in phosphate buffer. Proteins were eluted with 250 mM imidazole in phosphate buffer. For insoluble proteins a denaturing protocol was performed by firstly incubating the bacterial cells in an 8M urea (pH 7.8) solution containing 20mM sodium phosphate and 500mM NaCl. The denatured cellular matter was incubated as before with Ni-NTA beads and non- specific bound proteins were removed with two washes in 4M Urea (first wash pH6; second wash pH5) solution containing 20mM Sodium Phosphate and 500 mM NaCl.
  • 8M urea pH 7.8
  • the denatured cellular matter was incubated as before with Ni-NTA beads and non- specific bound proteins were removed with two washes in 4M Urea (first wash pH6; second wash pH5) solution containing 20mM Sodium Phos
  • Proteins were eluted with 4M Urea (pH3) in a solution containing 20mM Sodium Phosphate buffer and 500mM NaCl. Proteins were renatured after purification in 50mM Sodium Phosphate solution and 500mM NaCl. The purity and size of the expressed and purified proteins were assessed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were quantified using Bradford reagent (Bio-Rad).
  • Enzyme-linked immunosorbent assays using S. Typhi protein antigens ELISAs to detect antigen specific IgM in human plasma samples were performed as described by Karkey et al (2013) PLos Negl Trop Dis 7:e2391 with 12 purified protein antigens (Seq ID Nos: 1 to 12) and S. Typhi Vi polysaccharide antigen (provided by Sclavo Berhing Vaccines Institute for Global Health, Siena, Italy).
  • 96 well flat-bottom ELISA plates (Nunc 2404, Thermo Scientific) were coated overnight with ⁇ ⁇ per well of the various antigens (final concentrations; 7 ⁇ 1/ ⁇ 1 of protein antigens and ⁇ g/ml for the Vi polysaccharide antigen in 50mM Carbonate Bicarbonate buffer). Coated plates were washed and blocked with 5% fat free milk solution in phosphate buffered saline. After 2 hours of blocking, plates were washed and incubated with ⁇ ⁇ (per well) of a 1 :200 dilution of plasma at ambient temperature for 2 hours.
  • a geometric mean optical density was calculated to summarize the IgM response to the S. Typhi antigens in each arm of validation group, including the negative reference population samples (healthy controls and other confirmed febrile infections) and the positive reference population (febrile patients confirmed to be infected with S. Typhi).
  • the Wilcoxon signed-rank test was used to test the null hypothesis; no difference in optical densities between the patient groups. Spearman's rho was used to investigate potential correlations between IgM antibody responses against the various antigens.
  • Receiver operating characteristic (ROC) curves were used to determine the optimal cut-off and the specificity and sensitivity of the various antigens.
  • a performance estimation of more than one antigen combination was evaluated using Support Vector Machine (SVM).
  • SVM Support Vector Machine
  • a SVM is a supervised learning model that analyses data for classification and regression analysis using a training and test data set. In this study this was used to investigate the performance of combining multiple antigens for serological testing. All analyses were performed with R software (version 3.3. 1 ; R Foundation for Statistical Computing). All confidence intervals (CIs) are reported 2- sided at the 95% intervals; all other significant testing were performed 2-sided with a significance level of p ⁇ 0.05.
  • Detectable IgM was measured against all twelve of the purified S. Typhi proteins and the Vi polysaccharide in all of the 89 samples subjected to ELISAs.
  • the IgM titers were compared from each of the antigens individually to assess the performance of the antigens and to identify potential correlations between potential serological targets. It was found that early IgM responses against the majority of the novel S. Typhi protein antigens, with the exception of STY 1522 (rho ⁇ 0.7), were highly correlated with one other (rho>0.8). Notably, the IgM response to the protein antigens correlated only weakly to those directed against the Vi polysaccharide (rho ⁇ 0.6). A summary of these data for the antigens encoded by STY452, STY 1086, STY3208, STY 1886 and the Vi antigen are shown in Figure 5. The diagnostic potentiation of IgM against Salmonella Typhi antigens
  • IgM The diagnostic potential of IgM directed at the purified antigens for identifying patients with typhoid fever was considered. Encouragingly, IgM against all twelve of the protein antigens and the Vi polysaccharide was significantly elevated in the plasma of the typhoid patients in comparison to the healthy controls (p ⁇ 0.05) ( Figure 6a and 6b). Furthermore, there was a significant differentiation in the plasma IgM titers between the typhoid patients and those with febrile disease with an alternative confirmed etiology with all antigens with the exception of STY 1522 (/? ⁇ 0.05)
  • the IgM responses against each of the antigens generated a set of continuous data that was used to generate receiver operating characteristic (ROC) curves to optimize the index cut off value .
  • the defined cut-off values of the thirteen antigens corresponded with a range of specificities between 0.58 and 0.84 and sensitivities ranging from 0.50 to 0.84; areas under the ROC curve (AUC) ranged from 0.7 to 0.85.
  • AUC areas under the ROC curve
  • none of the antigens demonstrated specificities or sensitivities >0.8.
  • Vi, STY4359 and STY 1886 were the three antigens with the greatest serodiagnostic capacity in discriminating typhoid cases from afebrile controls and other infections.
  • the specificities and sensitivities for identifying typhoid patients by IgM titers against Vi, STY4539 and STY 1886 were 0.8, 0.68; 0.82, 0.62 and 0.82, 0.62, respectively.
  • the AUCs were 0.835 (95%CI: 0.71 1 , 0.958), 0.767 (95%CI: 0.612, 0.921) and 0.767 (95%CI: 0.62, 0.913).
  • SVM support vector machine
  • IgM against Vi contributed to all of the combinations, while STY 1703, STY 1886 and STY4539 were present in more than half of the SVM combinations. The remaining nine antigens contributed to at least one combination that gave specificities and sensitivities >0.8. These results demonstrated that, in the majority of examples, a combination of up to four antigens was directly associated with an increased performance of the IgM serology. However, the best performing antigens for the identification of typhoid patients by IgM were Vi in combination with either STY 1703 or STY 1886 (Table 3 and Table 4, Figures 3 and 4). Identifying typhoid cases in patients with undiagnosed febrile disease
  • the PilL protein has been shown to be induced in human derived macrophages, and the type IV pili to which it is associated facilitates entry into host intestine epithelial cells.
  • CdtB encoded by STY 1886, is one of the two A sub-units of typhoid toxin, an AB type toxin.
  • Typhoid toxin is a virulence-associated factor of S. Typhi, which is currently thought to be associated with the early symptoms of typhoid fever. This data confirms that this component of typhoid toxin is immunogenic and may be important biomarker of acute typhoid.

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Abstract

L'invention concerne un procédé de détermination de l'état typhoïde d'un sujet, consistant à : a) obtenir un échantillon fourni par un sujet ; b) examiner l'échantillon à la recherche de la présence d'anticorps au produit d'expression d'un ou de plusieurs gènes sélectionnés dans le groupe constitué par STY0452 (SEQ ID NO : 1), STY0796 (SEQ ID NO : 2). STY1086 (SEQ ID NO : 3), STY1372 (SEQ ID NO : 4), STY1612 (SEQ ID NO : 5), STY4539 (SEQ ID NO : 6), STY1522 (SEQ ID NO : 7), STY1703 (SEQ ID NO : 8), STY1767 (SEQ ID NO : 9), STY1886 (SEQ ID NO : 10), STY3208 (SEQ ID NO : 11) et STY4910 (SEQ ID NO : 12) ; c) examiner l'échantillon à la recherche de la présence d'anticorps au polysaccharide Vi ; et d) déterminer si les taux d'anticorps déterminés aux étapes (b) et (c) sont un diagnostic de la fièvre typhoïde.
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