Borrelia burgdorferi sensu lato

B. burgdorferi s.l., the causative agent of Lyme Borreliosis, is a Gram-negative corkscrew shaped, microaerophilic bacterium of the family of Spirochaetaceae.

B. burgdorferi was first described in 1982 by W. Burgdorfer (31). In Europe, there are at least three species pathogenic for humans (B. burgdorferi sensu stricto (B. burgdorferi s.s.), B. afzelii and B. garinii). B. valaisiana might also be pathogenic for humans, as suggested by positive PCR results obtained from a skin biopsy (300). The pathogen is transmitted into man by hard ticks (Ixodidae). At the site of the tick bite the infection

starts primarily with a local skin infection (Erythema migrans (EM)), then the spirochetes disseminate into the whole body. They can persist for years if untreated and may result in a range of clinical symptoms such as arthritis, neurological disorders, skin manifestations and arrhythmia (333). There are studies which showed an indirect evidence for the association of B. garinii with neurological symptoms (79), while infections with B. burgdorferi s.s. and B. afzelii tend to lead to arthritic symptoms (356) and cutaneous manifestations, respectively (35).

According to the Centre of Disease Control and Prevention, Lyme Borreliosis (LB) accounts for 95% of all reported vector-borne diseases in the United States and incidence estimations in endemic areas range between 16 and 140 / 100,000 inhabitants per year (161, 377). Individuals with a high risk of tick exposure (forest workers) frequently have significantly increased antibody titers against the pathogen (262, 375).

At present, the diagnosis of LB is made on the basis of the clinical picture and serological tests (102). For serodiagnosis a combination of a screening enzyme-linked immunosorbent assay (ELISA) as first step and a Western blot for confirmation as second step are recommended (44). In Western blot analyses antibodies against individual Borrelia antigens, which have been separated by gel electrophoresis, can be detected. However, a positive serological result without any clinical symptoms is not sufficient for the diagnosis of LB. Other than serological methods for detection of the pathogen like cultivation of Borrelia from patients’ specimen is difficult, since the pathogen occurs at low number in infected tissue and is difficult to cultivate due to long doubling time and the need of complex media (12, 61, 242, 322). Only for patients presenting an EM the cultivation of Borrelia or the direct detection via PCR from skin biopsies is a suitable method (203, 295, 322). But all these methods are confined to their special indications.

The interpretation, especially of Western blot bands and their intensity, is difficult, labor-intensive and not standardized (160). Many tests use crude extracts from the cultured Borrelia (different strains, different antigen preparations), which leads to further problems. One major problem is cross reactions with antibodies against other bacteria like Treponema, Ehrlichia or Epstein-Barr-virus and furthermore Borrelia proteins which are expressed only under human immune pressure are missed in these tests. In summary, the tests available are not standardized and the results of different test are not comparable due to these problems.

Commercial serological tests or in-house produced ones can not distinguish between active and inactive infection, because the IgG and IgM antibodies remain detectable over years at high levels, even after a successful antibiotic treatment (333). There is a hope that recombinant antigens might overcome these limitations.

Recombinant antigens would probably contribute to improve specificity and sensitivity of the serodiagnosis of LB. They would offer the advantage of easier identification of the bands in the immunoblot, since specific antigens can be selected, antigens from different strains can be combined, the test would be standardizable and the missing antigens could be added (378). Furthermore, by the use of truncated proteins cross reactions can be avoided. In the last years, there were many efforts in the identification of recombinant antigens and many different antigens were reported (118, 119, 142, 144, 203, 217, 232, 233, 277, 278). The first test with promising results was reported by Gomez et al. (118). They used chimeric recombinant proteins of outer surface protein A (OspA), p93, OspB, OspC and p41. The recombinant VlsE seems to be another very promising and useful antigen (142, 217, 233). In EM patients a higher sensitivity than those obtained with commercial ELISA was reached with the recombinant antigen BBK32 (143, 203). Despite the advantages of recombinant antigens and identical specificity, the recombinant blot has not yet shown the equal sensitivity of the whole-cell lysate blot. At present, the diagnosis is still far away from

the standardized serological test with high sensitivity and specificity. Further recombinant antigens will be needed to succeed.

LB is treated with antibiotics, but results are poor for treatments at late stages of the disease (220, 269, 334, 337, 338, 384). The difficulties in treatment of late stages and the high sero-prevalence of LB (268) call for a vaccination against Borrelia.

Immunizations with several different recombinant B. burgdorferi s.l. proteins have been performed. Immunization with OspA was found to be safe and effective in a large clinical trial in the United States (335). The vaccine efficacy in preventing clinical LB was 49% after two doses of the vaccine and 76% after three injections (327). The vaccine was approved by the Food and Drug Administration in 1998, but in 2002 the vaccine was withdrawn from the market. During this time, the vaccine was not tested in Europe, due to the presence of three B. burgdorferi species (in the United States only B. burgdorferi s.s. is found) resulting in heterogeneity of the OspA protein. The OspA vaccine gives rise to the hope that a combination of different recombinant proteins can lead to the development of a safe and worldwide usable vaccine.