Correlation of Culture Positivity, PCR Positivity, and Burden of Borrelia burgdorferi Sensu Lato in Skin Samples of Erythema Migrans Patients with Clinical Findings.

BACKGROUND: Limited data are available regarding the relationship of Borrelia burden in skin of patients with erythema migrans (EM) and the disease course and post-treatment outcome. METHODS: We studied 121 adult patients with EM in whom skin biopsy specimens were cultured and analyzed by quantitative PCR for the presence of Borreliae. Evaluation of clinical and microbiological findings were conducted at the baseline visit, and 14 days, 2, 6, and 12 months after treatment with either amoxicillin or cefuroxime axetil. RESULTS: In 94/121 (77.7%) patients Borrelia was detected in skin samples by PCR testing and 65/118 (55.1%) patients had positive skin culture result (96.8% B. afzelii, 3.2% B. garinii). Borrelia culture and PCR results correlated significantly with the presence of central clearing and EM size, while Borrelia burden correlated significantly with central clearing, EM size, and presence of newly developed or worsened symptoms since EM onset, with no other known medical explanation (new or increased symptoms, NOIS). In addition, the logistic regression model for repeated measurements adjusted for time from inclusion, indicated higher Borrelia burden was a risk factor for incomplete response (defined as NOIS and/or persistence of EM beyond 14 days and/or occurrence of new objective signs of Lyme borreliosis). The estimated association between PCR positivity and unfavorable outcome was large but not statistically significant, while no corresponding relationship was observed for culture positivity. CONCLUSIONS: Higher Borrelia burden in EM skin samples was associated with more frequent central clearing and larger EM lesions at presentation, and with a higher chance of incomplete response.

A Novel multivalent OspA vaccine against Lyme borreliosis is safe and immunogenic in an adult population previously infected with Borrelia burgdorferi sensu lato.

Lyme borreliosis (LB) patients who recover, as well as previously infected asymptomatic individuals, remain vulnerable to reinfection with Borrelia burgdorferi sensu lato. There is limited information available about the use of OspA vaccines in this population. In this study, a randomized double-blind phase I/II trial was performed to investigate the safety and immunogenicity of a novel multivalent OspA vaccine in healthy adults who were either seronegative or seropositive for previous B. burgdorferi sensu lato infection. The participants received three monthly priming immunizations with either 30 µg or 60 µg alum-adjuvanted OspA antigen and a booster vaccination either 6 months or 9 to 12 months after the first immunization. The antibody responses to the six OspA serotypes included in the vaccine were evaluated. Adverse events were predominantly mild and transient and were similar in the seronegative and seropositive populations. Substantial enzyme-linked immunosorbent assay (ELISA) and surface-binding antibody responses against all six OspA antigens were induced after the primary immunization schedule in both populations, and they were substantially increased with both booster schedules. The antibody responses induced by the two doses were similar in the seronegative population, but there was a significant dose response in the seropositive population. These data indicate that the novel multivalent OspA vaccine is well tolerated and immunogenic in individuals previously infected with B. burgdorferi sensu lato. (This study is registered at ClinicalTrials.gov under registration no. NCT01504347.).

Evaluation of OspA vaccination-induced serological correlates of protection against Lyme borreliosis in a mouse model.

BACKGROUND: For clinical development of a novel multivalent OspA vaccine against Lyme borreliosis, serological assays are required which can be used to establish immune correlates of protection against infection with Borrelia. METHODS: Four assays (an OspA IgG ELISA, a competitive inhibition (CI) ELISA, a Borrelia surface-binding (SB) assay and a Borrelia killing assay) were used to evaluate the correlation between immune responses induced by rOspA 1/2 (a chimeric immunogen containing protective epitopes from OspA serotypes 1 and 2), and protective immunity against infection by B. burgdorferi s.s. (OspA-1) and B. afzelii (OspA-2). Mice were immunized with OspA 1/2 doses ranging from 0.3 ng to 100 ng, to induce a range of OspA antibody titers, and exposed to needle challenge with B. burgdorferi s.s. or tick challenge with B. afzelii. Receiver operator characteristics (ROC) curves were constructed for each assay, and the area under the curve (AUC), sensitivity, specificity and Youden Index were calculated. Potential cutoff antibody titers which could be used as correlates of vaccine-induced protection were derived from the maximum Youden Index. RESULTS: Immunization with OspA-1/2 provided dose-dependent protection against infection with B. burgdorferi s.s. and B. afzelii. Antibody responses detected by all four assays were highly significantly correlated with protection from infection by either B. burgdorferi s.s. (p<0.0001 to 0.0062) or B. afzelii (p<0.0001). ROC analyses of the diagnostic effectiveness of each assay showed the AUC to range between 0.95 and 0.79, demonstrating that all assays distinguish well between infected and non-infected animals. Based on sensitivity, specificity and AUC, the OspA IgG ELISA and SB assays best discriminated between infected and non-infected animals. CONCLUSIONS: All four assays differentiate well between Borrelia-infected and non-infected animals. The relatively simple, high throughput IgG ELISA would be suitable to establish immune correlates of protection for the novel OspA vaccine in clinical trials.

Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR.

B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB) globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM) lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive) and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8%) and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (P<0.001). The quantitative data also enabled relationships between Borrelia burden and patient symptoms to be evaluated. The bacterial load was significantly higher among patients with systemic symptoms than without (P = 0.02) and was significantly higher for biopsies retrieved from patients with EM lesions with central clearing (P<0.001). 16S copy numbers were moderately lower in samples from patients reporting a history of LB (P = 0.10). This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii-infected patients and Borrelia burden.

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.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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). INTERPRETATION: 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. FUNDING: 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.

Wide distribution of a high-virulence Borrelia burgdorferi clone in Europe and North America.

The A and B clones of Borrelia burgdorferi sensu stricto, distinguished by outer surface protein C (ospC) gene sequences, are commonly associated with disseminated Lyme disease. To resolve phylogenetic relationships among isolates, we sequenced 68 isolates from Europe and North America at 1 chromosomal locus (16S-23S ribosomal RNA spacer) and 3 plasmid loci (ospC,dbpA, and BBD14). The ospC-A clone appeared to be highly prevalent on both continents, and isolates of this clone were uniform in DNA sequences, which suggests a recent trans-oceanic migration. The genetic homogeneity of ospC-A isolates was confirmed by sequences at 6 additional chromosomal housekeeping loci (gap, alr, glpA, xylB, ackA, and tgt). In contrast, the ospC-B group consists of genotypes distinct to each continent, indicating geographic isolation. We conclude that the ospC-A clone has dispersed rapidly and widely in the recent past. The spread of the ospC-A clone may have contributed, and likely continues to contribute, to the rise of Lyme disease incidence.

Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses.

The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.

Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.

To evaluate multilocus sequence analysis (MLSA) for taxonomic purposes in the delineation of species within Borrelia burgdorferi sensu lato, seven relevant loci of various strains for which extensive DNA-DNA reassociation data were available were sequenced. MLSA delineation proved to be fully concordant with conventional methods. Our analysis confirmed the delineation of a novel species, Borrelia spielmanii sp. nov., previously known as 'Borrelia spielmani' Richter et al. 2004, with strain PC-Eq17N5T (=DSM 16813T = CIP 108855T) as the type strain.

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses.

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.

Gene gun bombardment with gold particles displays a particular Th2-promoting signal that over-rules the Th1-inducing effect of immunostimulatory CpG motifs in DNA vaccines.

The mode of administering a DNA vaccine can influence the type of immune response induced by the vaccine. For instance, application of a DNA vaccine by gene gun typically induces a Th2-type reaction, whereas needle inoculation triggers a Th1 response. It has been proposed that the approximately 100-fold difference in the amount of DNA administered by these two methods is the critical factor determining whether a Th1 or a Th2 response is made. To test this hypothesis, BALB/c mice were immunized with two plasmid DNA constructs encoding different proteins (OspC/ZS7 of Borrelia burgdorferi and Bet v 1a, the major birch pollen allergen). Both vaccines were applied by needle and/or by gene gun immunization at the same and at different sites of injection. An analysis of the IgG subclass distribution and measurement of IFN-gamma after antigen-specific lymphoproliferation does not support the widely accepted view that Th2-type immunity induced by gene gun application is solely due to the low amount of injected plasmid DNA thus falling below the critical concentration of CpG motifs necessary for Th1-induction. Furthermore, the data also indicate a strong and even systemic adjuvant effect of the gene gun shot itself.