scholarly journals Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease

2014 ◽  
Vol 21 (5) ◽  
pp. 704-711 ◽  
Author(s):  
Giacomo Signorino ◽  
Paul M. Arnaboldi ◽  
Mary M. Petzke ◽  
Raymond J. Dattwyler
Keyword(s):  
Lyme Disease ◽  
Disease Patient ◽  
Laboratory Diagnosis ◽  
Etiologic Agent ◽  
Early Infection ◽  
Protein Antigens ◽  
Content Type ◽  
Linear Epitopes ◽  
Bacterial Lysates ◽  
Oligopeptide Permease

ABSTRACTLaboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agentBorrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number ofB. burgdorferiantigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique toB. burgdorferias well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique toB. burgdorferias antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during earlyB. burgdorferiinfection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenicBorreliaspecies responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease.

scholarly journals Decorin Binding Proteins A and B in the Serodiagnosis of Lyme Disease in North America

2014 ◽  
Vol 21 (10) ◽  
pp. 1426-1436 ◽  
Author(s):  
Paul M. Arnaboldi ◽  
Mariya Sambir ◽  
Raymond J. Dattwyler
Keyword(s):  
Lyme Disease ◽  
Protein A ◽  
Laboratory Diagnosis ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Antigens ◽  
Content Type ◽  
Linear Epitopes ◽  
The Individual

ABSTRACTThe laboratory diagnosis of Lyme disease is based upon the detection of antibodies generated againstBorrelia burgdorferiusing a two-tier assay, typically consisting of an enzyme-linked immunosorbent assay (ELISA), followed by a Western blot. This system, put into place to address the nonspecificity associated with standalone first-tier assays, is insensitive for diagnosing early infection, when most people seek care. The use of bacterial lysates or whole-protein antigens as first-tier assay targets contributes to nonspecificity due, in part, to the presence of cross-reactive epitopes that are also found in other bacteria. This precludes their use as sensitive standalone assays. The use of peptides containing linear epitopes that are highly specific forB. burgdorferioffers a method for reducing this cross-reactivity. In the present study, we mapped the linear epitopes of the prominently expressedBorreliaadhesins decorin binding protein A (DbpA) and DbpB. We identified several epitopes in each protein that were highly conserved among North American strains ofB. burgdorferi, and we screened peptides containing specific epitopes using serum panels from early and late Lyme disease patients. The individual peptides primarily detected IgM but not IgG, while the proteins efficiently detected both IgM and IgG. While no individual peptide demonstrated better utility for antibody detection than its respective whole protein, an assay containing a combination of a DbpA and a DbpB peptide adequately detected both IgM and IgG, accurately identifying 87.5% (84/96) of the early Lyme disease patients and 80.0% (16/20) of the late Lyme disease patients.

scholarly journals A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against LethalStreptococcus pneumoniaeChallenge

2018 ◽  
Vol 87 (3) ◽  
Author(s):  
Win-Yan Chan ◽  
Claire Entwisle ◽  
Giuseppe Ercoli ◽  
Elise Ramos-Sevillano ◽  
Ann McIlgorm ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Rabbit Serum ◽  
Protein Antigens ◽  
Content Type ◽  
Multiple Antigen ◽  
Bacterial Lysates ◽  
Chromatography Step

ABSTRACTCurrent vaccination againstStreptococcus pneumoniaeuses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared fromS. pneumoniaeTIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains ofS. pneumoniaewere opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologousS. pneumoniaestrains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection againstS. pneumoniae.

scholarly journals Outer Surface Protein C Peptide Derived from Borrelia burgdorferi Sensu Stricto as a Target for Serodiagnosis of Early Lyme Disease

2013 ◽  
Vol 20 (4) ◽  
pp. 474-481 ◽  
Author(s):  
Paul M. Arnaboldi ◽  
Rudra Seedarnee ◽  
Mariya Sambir ◽  
Steven M. Callister ◽  
Josephine A. Imparato ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Bacterial Species ◽  
Erythema Migrans ◽  
Early Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Attractive Alternative ◽  
Serum Samples ◽  
Peptide Epitope ◽  
Content Type

ABSTRACTCurrent serodiagnostic assays for Lyme disease are inadequate at detecting early infection due to poor sensitivity and nonspecificity that arise from the use of whole bacteria or bacterial proteins as assay targets; both targets contain epitopes that are cross-reactive with epitopes found in antigens of other bacterial species. Tests utilizing peptides that contain individual epitopes highly specific forBorrelia burgdorferias diagnostic targets are an attractive alternative to current assays. Using an overlapping peptide library, we mapped linear epitopes in OspC, a critical virulence factor ofB. burgdorferirequired for mammalian infection, and confirmed the results by enzyme-linked immunosorbent assay (ELISA). We identified a highly conserved 20-amino-acid peptide epitope, OspC1. Via ELISA, OspC1 detected specific IgM and/or IgG in 60 of 98 serum samples (62.1%) obtained from patients with erythema migrans (early Lyme disease) at the time of their initial presentation. By comparison, the commercially available OspC peptide PepC10 detected antibody in only 48 of 98 serum samples (49.0%). In addition, OspC1 generated fewer false-positive results among negative healthy and diseased (rheumatoid arthritis and positive Rapid Plasma Reagin [RPR+] test result) control populations than did PepC10. Both highly specific and more sensitive than currently available OspC peptides, OspC1 could have value as a component of a multipeptide Lyme disease serological assay with significantly improved capabilities for the diagnosis of early infection.

scholarly journals Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

2012 ◽  
Vol 19 (4) ◽  
pp. 527-535 ◽  
Author(s):  
Bettina Wagner ◽  
Heather Freer ◽  
Alicia Rollins ◽  
David Garcia-Tapia ◽  
Hollis N. Erb ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
The United States ◽  
Sensu Stricto ◽  
Antibody Responses ◽  
Surface Proteins ◽  
Early Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Antibody Levels

ABSTRACTLyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

scholarly journals A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use

2019 ◽  
Vol 57 (12) ◽  
Author(s):  
Siddarth Arumugam ◽  
Samiksha Nayak ◽  
Taylor Williams ◽  
Francesco Serra di Santa Maria ◽  
Mariana Soares Guedes ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Confidence Interval ◽  
Point Of Care ◽  
Laboratory Diagnosis ◽  
Lyme Arthritis ◽  
Disease Stage ◽  
Serologic Test ◽  
Content Type ◽  
Disease Stages ◽  
Late Stages

ABSTRACT Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.

scholarly journals Cross-Reactive Epitopes in Borrelia burgdorferi p66

2015 ◽  
Vol 22 (7) ◽  
pp. 840-843 ◽  
Author(s):  
Paul M. Arnaboldi ◽  
Raymond J. Dattwyler
Keyword(s):  
Lyme Disease ◽  
Membrane Protein ◽  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Epitope Mapping ◽  
Serum Antibody ◽  
Content Type ◽  
Linear Epitopes

ABSTRACTEpitope mapping of the p66 outer membrane protein ofBorrelia burgdorferirevealed that the protein contains numerous cross-reactive linear epitopes recognized by serum antibody in the majority of individuals tested, regardless of Lyme disease history, limiting the usefulness of this antigen in Lyme disease serodiagnostic assays.

scholarly journals Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

2013 ◽  
Vol 20 (3) ◽  
pp. 350-357 ◽  
Author(s):  
Micah D. Halpern ◽  
Sunny Jain ◽  
Mollie W. Jewett
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Host Response ◽  
Magnetic Beads ◽  
Disease Patient ◽  
Rank Correlation ◽  
Protein Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type

ABSTRACTLyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection ofBorrelia burgdorferiinfection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinantin vivo-expressedB. burgdorferiantigens for objective detection of a host immune response toB. burgdorferiinfection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinantB. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (Cq) value for each sample minus the mean backgroundCqplus 3 standard deviations (ΔCq) being 4 to 10, whereas ΔCqwas 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895,P< 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using theB. burgdorferiVlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes toB. burgdorferiinfection.

scholarly journals Linear B Cell Epitopes Derived from the Multifunctional Surface Lipoprotein BBK32 as Targets for the Serodiagnosis of Lyme Disease

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Christina Toumanios ◽  
Lauren Prisco ◽  
Raymond J. Dattwyler ◽  
Paul M. Arnaboldi
Keyword(s):  
Amino Acids ◽  
Lyme Disease ◽  
B Cell ◽  
Erythema Migrans ◽  
Antibody Binding ◽  
Early Infection ◽  
Early Disease ◽  
B Cell Epitopes ◽  
Content Type ◽  
Linear B

ABSTRACTBBK32 is a multifunctional surface lipoprotein expressed byBorrelia burgdorferisensu lato,the causative agent of Lyme disease. Previous studies suggested that BBK32 could be a sensitive antigen target of new, more effective, serodiagnostic assays for the laboratory diagnosis of Lyme disease. However, nonspecific antibody binding to full-length BBK32 has hampered its use as a target in clinical assays. Specificity can be improved by the use of peptides composed of linear B cell epitopes that are unique toB. burgdorferi, eliminating cross-reactive epitopes that bind to antibodies generated by non-B. burgdorferiantigens. In this study, we identified linear B cell epitopes in 2 regions, BBK32 amino acids 16 to 30 [BBK32(16–30)] and BBK32 amino acids 51 to 80 [BBK32(51–80)], by probing overlapping peptide libraries of BBK32 with serum from patients with early Lyme disease. We screened synthetic peptides containing these epitopes using a large panel of serum (n = 355) obtained from patients with erythema migrans lesions (early Lyme disease), Lyme arthritis, syphilis, rheumatoid arthritis, or healthy volunteers. BBK32(16–30) demonstrated a nearly universal antibody binding in serum from all patients, indicating that regions of BBK32 are highly cross-reactive. BBK32(51–80) was less cross-reactive, being able to distinguish serum from Lyme disease patients from control patient serum; however, an unacceptable level of antibody binding was still observed in control samples, resulting in a reduced specificity (94.7%). These results indicate that BBK32 contains cross-reactive epitopes that make it a poor antigen target for inclusion in a serodiagnostic assay for Lyme disease and highlight the difficulties in identifying highly sensitive and specific seroassay targets.IMPORTANCELyme disease is an infectious disease that has the potential to cause significant morbidity with damage to nervous and musculoskeletal systems if left untreated. Appropriate antibiotic treatment during early infection prevents disease progression. Unfortunately, currently available diagnostics are suboptimal in the detection of early disease. The inability to confirmBorreliainfection using laboratory methods during early disease is, in part, responsible for much of the controversy surrounding Lyme disease today. As a result, there has been significant investment in the identification of new antigen targets to generate diagnostic assays that are more sensitive for the detection of early infection. The importance of our research is that in our evaluation of BBK32, an antigen that was previously identified as a promising target for use in serodiagnostics, we found a high degree of cross-reactivity that could compromise the specificity of assays that utilize this antigen, leading to false-positive diagnoses.

scholarly journals Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease

2015 ◽  
Vol 53 (12) ◽  
pp. 3834-3841 ◽  
Author(s):  
Lauren J. Lahey ◽  
Michael W. Panas ◽  
Rong Mao ◽  
Michelle Delanoy ◽  
John J. Flanagan ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Early Stage ◽  
Erythema Migrans ◽  
Disease Patient ◽  
High Specificity ◽  
The United States ◽  
Surface Proteins ◽  
Current Standard ◽  
Content Type

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies againstBorrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62B. burgdorferisurface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively;P< 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting earlyB. burgdorferiinfection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.

scholarly journals Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

2014 ◽  
Vol 21 (8) ◽  
pp. 1094-1105 ◽  
Author(s):  
Micah D. Halpern ◽  
Claudia R. Molins ◽  
Martin Schriefer ◽  
Mollie W. Jewett
Keyword(s):  
Lyme Disease ◽  
Effective Means ◽  
Disease Patient ◽  
Healthy Population ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Content Type ◽  
Laboratory Confirmation ◽  
Coefficients Of Variation ◽  
Objective Detection

ABSTRACTA serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detectingBorrelia burgdorferiantibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n= 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n= 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n= 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n= 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies againstB. burgdorferi.

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