RESUMEN
Lyme disease (LD) results from the most prevalent tick-borne infection in North America, with over 476,000 estimated cases annually. The disease is caused by Borrelia burgdorferi (Bb) sensu lato which transmits through the bite of Ixodid ticks. Most cases treated soon after infection are resolved by a short course of oral antibiotics. However, 10-20% of patients experience chronic symptoms because of delayed or incomplete treatment, a condition called Post-Treatment Lyme Disease (PTLD). Some Bb persists in PTLD patients after the initial course of antibiotics and an effective treatment to eradicate the persistent Bb is needed. Other organisms that cause persistent infections, such as M. tuberculosis, are cleared using a combination of therapies rather than monotherapy. A group of Food and Drug Administration (FDA)-approved drugs previously shown to be efficacious against Bb in vitro were used in monotherapy or in combination in mice infected with Bb. Different methods of detection were used to assess the efficacy of the treatments in the infected mice including culture, xenodiagnosis, and molecular techniques. None of the monotherapies eradicated persistent Bb. However, 4 dual combinations (doxycycline + ceftriaxone, dapsone + rifampicin, dapsone + clofazimine, doxycycline + cefotaxime) and 3 triple combinations (doxycycline + ceftriaxone+ carbomycin, doxycycline + cefotaxime+ loratadine, dapsone+ rifampicin+ clofazimine) eradicated persistent Bb infections. These results suggest that combination therapy should be investigated in preclinical studies for treating human Lyme disease.
RESUMEN
Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales/farmacología , Borrelia burgdorferi , Enfermedad de Lyme , Sustitución de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Modelos Animales de Enfermedad , Humanos , Lipoproteínas/genética , Lipoproteínas/inmunología , Enfermedad de Lyme/tratamiento farmacológico , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/transmisión , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Mutación Missense , Garrapatas/inmunología , Garrapatas/microbiologíaRESUMEN
The identification of microbial biomarkers is critical for the diagnosis of a disease early during infection. However, the identification of reliable biomarkers is often hampered by a low concentration of microbes or biomarkers within host fluids or tissues. We have outlined a multi-platform strategy to assess microbial biomarkers that can be consistently detected in host samples, using Borrelia burgdorferi, the causative agent of Lyme disease, as an example. Key aspects of the strategy include the selection of a macaque model of human disease, in vivo Microbial Antigen Discovery (InMAD), and proteomic methods that include microbial biomarker enrichment within samples to identify secreted proteins circulating during infection. Using the described strategy, we have identified 6 biomarkers from multiple samples. In addition, the temporal antibody response to select bacterial antigens was mapped. By integrating biomarkers identified from early infection with temporal patterns of expression, the described platform allows for the data driven selection of diagnostic targets.
Asunto(s)
Biomarcadores , Borrelia burgdorferi/aislamiento & purificación , Enfermedad de Lyme/diagnóstico , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Técnicas Bacteriológicas , Biomarcadores/sangre , Biomarcadores/orina , Borrelia burgdorferi/inmunología , Diagnóstico Precoz , Humanos , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Macaca mulatta , Proteómica , Suero/química , Orina/químicaRESUMEN
The efficacy and accepted regimen of antibiotic treatment for Lyme disease has been a point of significant contention among physicians and patients. While experimental studies in animals have offered evidence of post-treatment persistence of Borrelia burgdorferi, variations in methodology, detection methods and limitations of the models have led to some uncertainty with respect to translation of these results to human infection. With all stages of clinical Lyme disease having previously been described in nonhuman primates, this animal model was selected in order to most closely mimic human infection and response to treatment. Rhesus macaques were inoculated with B. burgdorferi by tick bite and a portion were treated with recommended doses of doxycycline for 28 days at four months post-inoculation. Signs of infection, clinical pathology, and antibody responses to a set of five antigens were monitored throughout the ~1.2 year study. Persistence of B. burgdorferi was evaluated using xenodiagnosis, bioassays in mice, multiple methods of molecular detection, immunostaining with polyclonal and monoclonal antibodies and an in vivo culture system. Our results demonstrate host-dependent signs of infection and variation in antibody responses. In addition, we observed evidence of persistent, intact, metabolically-active B. burgdorferi after antibiotic treatment of disseminated infection and showed that persistence may not be reflected by maintenance of specific antibody production by the host.
Asunto(s)
Borrelia burgdorferi/fisiología , Ixodes/microbiología , Primates/parasitología , Animales , Ixodes/fisiología , Ratones , Mordeduras de GarrapatasRESUMEN
The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease.