OspA Antigen Research Articles

A portable immunosensor provides sensitive and rapid detection of Borrelia burgdorferi antigen in spiked blood

There are no assays for detecting B. burgdorferi antigen in blood of infected Lyme disease individuals. Here, we provide proof-of-principle evidence that we can quantify B. burgdorferi antigen in spiked blood using a portable smartphone-based fluorescence microscope that measures immunoagglutination on a paper microfluidic chip. We targeted B. burgdorferi OspA to develop a working prototype and added examples of two antigens (OspC and VlsE) that have diagnostic value for discrimination of Lyme disease stage. Using an extensively validated monoclonal antibody to OspA (LA-2), detection of OspA antigen had a broad linear range up to 100 pg/mL in 1% blood and the limit of detection (LOD) was 100 fg/mL (= 10 pg/mL in undiluted blood), which was 1000 times lower than our target of 10 ng/mL. Analysis of the two other targets was done using polyclonal and monoclonal antibodies. OspC antigen was detected at LOD 100 pg/mL (= 10 ng/mL of undiluted blood) and VlsE antigen was detected at LOD 1–10 pg/mL (= 0.1–1 ng/mL of undiluted blood). The method is accurate and was performed in 20 min from sample to answer. When optimized for detecting several B. burgdorferi antigens, this assay may differentiate active from past infections and facilitate diagnosis of Lyme disease in the initial weeks of infection, when antibody presence is typically below the threshold to be detected by serologic methods.

Detection of Borrelia burgdorferi Sensu-Lato-Specific Antibodies in Sera of Canine and Equine Origin-A Comparative Study with Two Line Immunoassays.

Lyme borreliosis is a vector-borne disease in humans and animals caused by bacteria from the Borrelia burgdorferi sensu lato complex (Bbsl). The possible transmission of Bbsl from companion animals to humans via ticks makes this disease important in terms of One Health approaches. Thus, early and accurate diagnosis and treatment are of utmost importance. Today's standard for the detection of specific antibodies against Bbsl is a two-tiered test system based on an ELISA for screening combined with a line immunoassay (LIA) for confirmation. In this study, 200 canine and 200 equine serum samples with known antibody status were tested with two different LIAs (A and B). Results were compared regarding sensitivity, specificity, the diagnostic outcome for dogs and horses, as well as operability of the test. The results for canine serum samples corresponded to 94.0%, making both LIAs a good choice for LB diagnostic in dogs. For equine serum samples, the agreement of both tests was 65.5%, displaying the challenge equine samples still provide in LB diagnostic. Major concerns were the interpretation of the OspA antigen (AG) signal and the use of unspecific (i.e., p100/p83) or too sensitive signals on the LIA. The operability of both LIAs was equally user-friendly. Regarding the tests' evaluation, the scanning process provided by LIA A was a major advantage considering the comparability of the tests.

Characterization of recombinant OspA in two different Borrelia vaccines with respect to immunological response and its relationship to functional parameters

BackgroundPrevention of Lyme disease in dogs in North America depends on effective vaccination against infection by the tick vector-born spirochete Borrelia burgdorferi. Most vaccines effectively prevent spirochete transmission to dogs during tick feeding based on immunization with the outer-surface lipoprotein A (OspA) of B. burgdorferi. More recently, vaccines containing additional OspC protein moieties have been introduced. These are designed to enhance protection by forming a second line of defense within the vertebrate host, where OspC expression replaces OspA as the dominant surface antigen. However, supportive data for demonstration of OspC mediated protection is still lacking. Since OspA immunogenicity is of paramount importance to protection against spirochete transmission; this study was designed to compare the immunogenicity of two commercially available vaccines against the Borrelia burgdorferi OspA. We further characterized OspA antigen fractions of these vaccines with respect to their biochemical and biophysical properties.ResultsTwo groups of beagle dogs (n = 9) were administered either: (1) a nonadjuvanted/monovalent, recombinant OspA vaccine (Recombitek® Lyme) or (2) an adjuvanted, recombinant OspA /OspC chimeric fusion vaccine (Vanguard® crLyme). The onset of the anti-OspA antibody response elicited by the nonadjuvanted/monovalent OspA vaccine was significantly earlier than that for the bivalent OspA /OspC vaccine and serum borreliacidal activity was significantly greater at all post-vaccination time points. As expected, only dogs inoculated with the bivalent OspA/OspC vaccine mounted a humoral anti-OspC response. However, only three out of nine dogs in that group had a positive response. Comparison of the OspA vaccine structures revealed that the OspA in the nonadjuvanted/monovalent vaccine was primarily in the lipidated form, eluting (SEC-HPLC) at a high molecular weight, suggestive of micelle formation. Conversely, the OspA moiety of the OspA/OspC vaccine was found to be nonlipidated and eluted as the monomeric protein.ConclusionsWe hypothesize that these structural differences may account for the superior immunogenicity of the nonadjuvanted monovalent recombinant OspA vaccine in dogs over the adjuvanted OspA fraction of the OspA/OspC vaccine.

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

ObjectivesPrompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB.MethodWe employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation.ResultsOspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7–30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10−6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e−15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.ConclusionsOspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0701-z) contains supplementary material, which is available to authorized users.

Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomised, dose-escalation phase 1/2 trial

Lyme borreliosis is caused by Borrelia burgdorferi sensu stricto in the USA and by several Borrelia species in Europe and Asia, but no human vaccine is available. We investigated the safety and immunogenicity of adjuvanted and non-adjuvanted vaccines containing protective epitopes from Borrelia species outer surface protein A (OspA) serotypes in healthy adults. Between March 1, 2011, and May 8, 2012, we did a double-blind, randomised, dose-escalation phase 1/2 study at four sites in Austria and Germany. Healthy adults aged 18-70 years who were seronegative for B. burgdorferi sensu lato were eligible for inclusion. Participants were recruited sequentially and randomly assigned to one of six study groups in equal ratios via an electronic data capture system. Participants and investigators were masked to group allocation. Participants received three vaccinations containing 30 μg, 60 μg, or 90 μg OspA antigen with or without an adjuvant, with intervals of 28 days, and a booster 9-12 months after the first immunisation. The coprimary endpoints were the frequency and severity of injection-site and systemic reactions within 7 days of each vaccination, and the antibody responses to OspA serotypes 1-6, as established by ELISA. This study is registered with ClinicalTrials.gov, number NCT01504347. 300 participants were randomly assigned: 151 to adjuvanted vaccines (50 to 30 μg, 51 to 60 μg, and 50 to 90 μg doses), and 149 to non-adjuvanted vaccines (50 to 30 μg, 49 to 60 μg, and 50 to 90 μg doses). Adverse reactions were predominantly mild, and no vaccine-related serious adverse events were reported. The risk of systemic reactions (risk ratio 0·54 [95% CI 0·41-0·70]; p<0·0001) and of moderate or severe systemic reactions (0·35 [0·13-0·92]; p=0·034) was significantly lower for adjuvanted than non-adjuvanted formulations. The 30 μg adjuvanted formulation had the best tolerability profile; only headache (five [10%, 95% CI 4-20] of 50), injection-site pain (16 [32%, 21-45]), and tenderness (17 [34%, 23-47]) affected more than 6% of patients. All doses and formulations induced substantial mean IgG antibody titres against OspA serotypes 1-6 after the first three vaccinations (range 6944-17,321) and booster (19,056-32,824) immunisations. The 30 μg adjuvanted formulation induced the highest antibody titres after the booster: range 26,143 (95% CI 18,906-36,151) to 42,381 (31,288-57,407). The novel multivalent OspA vaccine could be an effective intervention for prevention of Lyme borreliosis in Europe and the USA, and possibly worldwide. Larger confirmatory formulation studies will need to be done that include individuals seropositive for Borrelia burgdorferi sensu lato before placebo-controlled phase 3 efficacy studies can begin. Baxter.

A New Approach to a Lyme Disease Vaccine

A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borrelia afzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B. afzelii species was shown in a tick-challenge model using feral ticks. In both models, as little as .03 μg of antigen, when administered in a 2-dose immunization schedule with aluminum hydroxide as adjuvant, was sufficient to provide complete protection against the species targeted. This proof of principle study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines requiring fewer OspA antigens and suggests that this approach may facilitate the development of an OspA vaccine for global use.

Serum Reactivity against Borrelia burgdorferi OspA in Patients with Rheumatoid Arthritis

Lyme arthritis and rheumatoid arthritis share common clinical features and synovial histology. It is unclear whether they also share similar pathogenesis. Previous studies have shown that the severity and duration of Lyme arthritis correlate directly with serum concentrations of antibody against outer surface protein A (OspA) of the causative pathogen Borrelia burgdorferi. We tested the sera of 68 subjects with rheumatoid arthritis, 147 subjects with other autoimmune diseases, and 44 healthy subjects who had never had Lyme disease, as well as sera of 16 patients who had Lyme disease, for reactivity against the B. burgdorferi OspA protein. The sera of about a quarter of the rheumatoid arthritis patients and a 10th of the autoimmune disease and Lyme disease patients reacted against OspA antigen. Of 50 rheumatoid arthritis patients who could be evaluated for disease severity, a 28-joint count disease activity score of >2.6 was noted for 11 of 15 (73%) patients whose sera reacted against OspA antigen and 13 of 35 (37%; P < 0.05) whose sera were nonreactive. Serum reactivity against OspA antigen is associated with the pathogenesis of rheumatoid arthritis.

Structure-based Design of a Second-generation Lyme Disease Vaccine Based on a C-terminal Fragment of Borrelia burgdorferi OspA

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi elicits protective immunity that blocks transmission of Borrelia from the tick vector to the vaccinated animal, and thus has been a focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer beta-sheet. All anti-OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of using the C-terminal domain alone as a recombinant protein-based vaccine. The removal of ineffective parts from the OspA antigen may reduce side effects and lead to a safer vaccine. We prepared a C-terminal fragment of OspA by removing approximately 45% of residues from the N terminus. Although the fragment retained the native conformation and affinity to a protective antibody, its vaccine efficacy and conformational stability were significantly reduced with respect to full-length OspA. We successfully stabilized the fragment by replacing amino acid residues involved in buried salt-bridges with residues promoting hydrophobic interactions. The mutations promoted the vaccine efficacy of the redesigned fragment to a level comparable to that of the full-length protein, demonstrating the importance of the antigen stability for OspA's vaccine efficacy. Our strategy should be useful for further refining OspA-based vaccines and developing recombinant vaccines for other diseases.

Neuropathy and cognitive impairment following vaccination with the OspA protein of Borrelia burgdorferi.

Neurological syndromes that follow vaccination or infection are often attributed to autoimmune mechanisms. We report six patients who developed neuropathy or cognitive impairment, within several days to 2 months, following vaccination with the OspA antigen of Borrelia burgdorferi. Two of the patients developed cognitive impairment, one chronic inflammatory demyelinating polyneuropathy (CIDP), one multifocal motor neuropathy, one both cognitive impairment and CIDP, and one cognitive impairment and sensory axonal neuropathy. The patients with cognitive impairment had T2 hyperintense white matter lesions on magnetic resonance imaging. The similarity between the neurological sequelae observed in the OspA-vaccinated patients and those with chronic Lyme disease suggests a possible role for immune mechanisms in some of the manifestations of chronic Lyme disease that are resistant to antibiotic treatment.

Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine

In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti- B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector.

Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii

Sequence variability of the outer surface protein (Osp) A among Borrelia burgdorferi sl species suggests that a monovalent OspA vaccine may not protect against the various Borrelia present in Eurasia. Here, we confirmed that a monovalent recombinant OspA (rOspA) vaccine does not protect mice against Ixodes ricinus mediated infection with B. burgdorferi ss, Borrelia garinii and Borrelia afzelii. However, when mice were vaccinated with a cocktail of various rOspA from these three species, they were protected, and all challenge ticks that fed on them were cleared of their spirochetes. These results showed that a multiple OspA antigens vaccine, compatible with human use, was very efficient at protecting mice against B. burgdorferi ss, B. garinii, and B. afzelii.

Detection of Borrelia burgdorferi-specific antigen in antibody-negative cerebrospinal fluid in neurologic Lyme disease.

To determine the potential of detection in CSF of specific Borrelia burgdorferi antigen, OspA, as a marker of infection in neurologic Lyme disease and compare this with the detection of antibody. CSF from 83 neurologic patients in an area highly endemic for Lyme disease was examined prospectively for (1) OspA by antigen capture ELISA and Western blot employing monoclonal antibodies, and for (2) B burgdorferi antibodies by ELISA. Of the 35 of 83 (42%) patients who were positive for OspA antigen in their CSF, 15 (43%) were antigen positive despite being antibody-negative in CSF. Seven of these 15 (47%) had otherwise normal routine CSF analyses. Six of these 15 (40%) patients met strict CDC surveillance criteria for Lyme disease; four (27%) patients had seroconversion coincident with new neurologic problems; and three (20%) with characteristic syndromes for Lyme disease were seronegative, but had complexed antibody to B burgdorferi. The final two patients (13%) were seropositive and had unexplained neurologic problems not characteristic of Lyme disease. B burgdorferi antigen can be detected in CSF that is otherwise normal by conventional methodology, and can be present without positive CSF antibody. Since CSF antigen implies intrathecal seeding of the infection, the diagnosis of neurologic infection by B burgdorferi should not be excluded solely on the basis of normal routine CSF or negative CSF antibody analyses.

A murine IgG1 monoclonal antibody that binds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi.

A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi, has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii. The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA as well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi, has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii. The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA as well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.