Prevention of lyme disease: promising research or sisyphean task?

Borrelia burgdorferi sensu lato (Spirochaetes) is a group of at least 12 closely related species, some of which are responsible for chronic zoonotic infection that may cause Lyme disease. The only experimentally confirmed vector transmitting Borrelia to mammals is the Ixodes ticks. Borrelia is a highly adapted pathogen that can survive in the host organism in spite of the intense immune responses. Some patients have chronic long-lasting complications despite antibiotic therapy, probably due to adverse effects of the immune responses. A preventive vaccine against this bacterium has not been available due to the relatively broad spectrum and antigenic variability of Borrelia-surface lipoproteins and the different epitope recognition by experimental animals and humans. Although a human vaccine was marketed in the USA, it has been already pulled off the market. In addition, this vaccine was effective only in the USA, where the only pathogenic species is B. burgdorferi sensu stricto. Recent data indicate that a broadly effective vaccine will to be composed of a mixture of several antigens or multiple epitopes.

Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes.

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyses the oxygenation of cysteine to cysteine sulphinic acid leading to the production of sulphite, sulphate and taurine as the final metabolites of cysteine catabolism. Keratinolytic fungi secrete sulphite and sulphate to reduce disulphide bridges in host tissue keratin proteins as the first step of keratinolysis. In the present study, we describe the identification of cDNA, as well as expression and characterisation of recombinant CDO protein from Trichophyton mentagrophytes. The cDNA was amplified using primers designed on the basis of high conservancy CDO regions identified in other fungi. PCR product was cloned and sequenced. Recombinant CDO was expressed in Escherichia coli, and affinity purified and identified by matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS). Enzyme activity was assayed by monitoring the production of cysteine sulphinate using mass spectrometry. The Cdo cDNA encodes for a protein consisting of 219 amino acids. Recombinant CDO protein C-terminally fused with a His tag was purified by affinity chromatography. The CDO purified under native condition was proved to be enzymatically active. Protein identity was confirmed by MALDI-TOF MS. Comparison of cDNA sequence with those identified in other fungi revealed significant homology. Identification of T. mentagrophytes CDO provides indispensable tools for future studies of dermatophyte pathogenicity and development of new approaches for prevention and therapy.

Novel modification of growth medium enables efficient E. coli expression and simple purification of an endotoxin-free recombinant murine hsp70 protein.

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis.

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

DNA vaccines: are they still just a powerful tool for the future?

Vaccination is historically one of the most successful strategies for the prevention of infectious diseases. For safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. The antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. Differing ratios of various proportions between specific CD4+ and CD8+ T cell responses are essential for conferring the required protection in the case of individual vaccines. To stimulate both CD4+ and CD8+ T cells, the antigens must be processed and presented to both antigen-presentation pathways, MHC I and MHC II. Protein antigens delivered by vaccination are processed as extracellular antigens. However, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-DNA vaccination. In this overview, current knowledge of the host immune response to DNA vaccines is summarized in the introduction. The subsequent sections discuss techniques for enhancing DNA vaccine efficacy, such as DNA delivery to specific tissues, delivery of DNA to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. Finally, the prospects of DNA vaccination and ongoing clinical trials with various DNA vaccines are discussed.

Biological aspects of Lyme disease spirochetes: unique bacteria of the Borrelia burgdorferi species group.

BACKGROUND: Borrelia burgdorferi sensu lato is a group of at least twelve closely related species some of which are responsible for Lyme disease, the most frequent zoonosis in Europe and the USA. Many of the biological features of Borrelia are unique in prokaryotes and very interesting not only from the medical viewpoint but also from the view of molecular biology. METHODS: Relevant recent articles were searched using PubMed and Google search tools. RESULTS AND CONCLUSION: This is a review of the biological, genetic and physiological features of the spirochete species group, Borrelia burgdorferi sensu lato. In spite of a lot of recent articles focused on B. burgdorferi sensu lato, many features of Borrelia biology remain obscure. It is one of the main reasons for persisting problems with prevention, diagnosis and therapy of Lyme disease. The aim of the review is to summarize ongoing current knowledge into a lucid and comprehensible form.

Heat shock proteins in autoimmune diseases.

Heat shock proteins (hsp's) are among the most conserved proteins in evolution. They have been identified as important pathogen-related antigens as well as autoantigens suitable for construction of novel vaccines. The high evolutionary homology of hsp's has raised the question about the safety of such vaccines. Experimental and clinical observations have confirmed that hsp proteins are involved in the regulation of some autoimmune disease such as autoimmune arthritis, type 1 diabetes mellitus, atherosclerosis, multiple sclerosis, and other autoimmune reactions. It has been shown in experimental animals that some hsp proteins (especially hsp60, hsp70, and hsp10) can either induce or prevent autoimmune reactions depending on the circumstances. This article discusses the involvement of hsp proteins in the etiology of autoimmune diseases and it presents promising experimental data on the effects of immunization with hsp proteins in the prevention and therapy of autoimmune diseases.

Preparation and purification of recombinant outer surface protein A (rOspA) of Borrelia burgdorferi sensu stricto and Borrelia afzelii.

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitating disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recombinant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.

Isolation and purification of recombinant outer surface protein C (rOspC) of Borrelia burgdorferi sensu lato.

The aim of this work was isolation and purification of the major immunodominant protein, Outer surface protein C (OspC) of three members of the species group Borrelia burgdorferi, the causative agent of Lyme disease. Our aim was to obtain this protein in a quantity and purity sufficient for immunization of experimental animals. For optimalization of protein purification's yield we used immobilized metal ion affinity chromatography (IMAC) under different conditions. The greatest efficiency was achieved by using of HiTrap Chelating Column under native conditions.

Testing of the Biocan B inj. ad us. vet. vaccine and development of the new recombinant vaccine against canine borreliosis.

Verification of the efficacy of Biocan B inj. ad us. vet. (Bioveta, a.s.) was done by challenge testing. Ticks collected in the nature were used as natural vectors of the infection. Six beagles and two control ones were used in the test. Formation of outer surface protein A specific antibodies (OspA antibodies) and borrelia specific immonoglobulins (IgG) was measured by Western blot and EIA in the sera samples. The tissue samples were used for detection of borreliae by cultivation method and dark field microscopy (DFM). Formation of IgG antibodies and OspA antibodies after vaccination was observed. The maximum titer level of antibodies was reached between 21. and 49. day after vaccination and then slowly decreased. Presence of borreliae was detected only in skin biopsies of non-vaccinated dogs. The post mortem tissue samples showed presence of borreliae in all of the samples of the non-vaccinated dogs. The tissues of the vaccinated dogs were not infected with borreliae, except for two samples of dog with low titer levels of OspA antibodies. The development of the new vaccine is based on preparation of recombinant outer surface proteins (e.g. rOspA and rOspC) of B. afzelii, B. burgdorferi and B. garinii origin. Chosen recombinant proteins were successfully expressed in E. coli. The obtained purified proteins are currently being tested on laboratory BALB/c mice. Formation of specific antibodies against some recombinant proteins has been confirmed. These proteins are suitable candidates for preparation of a vaccine prototype and they will be subsequently used in challenge tests.

Frequency of 657del(5) mutation of the NBS1 gene in the Czech population by polymerase chain reaction with sequence specific primers.

Nijmegen breakage syndrome is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, immunodeficiency, radiosensitivity, and predisposition to lymphoid malignancy. A truncating deletion [657del(5)] in exon 6 of the nibrin NBS1 gene is the most frequent cause of the syndrome. Slavic populations carry this mutation in a high frequency. Here, we present polymerase chain reaction with sequence specific primers as a method for the detection of Slavic NBS1 mutation and confirm the high carrier frequency in the Czech population (combined frequency from both studies: 1/106, 95% CI = 1/331 to 1/46).

CC chemokine receptor 5 (CCR5) mRNA expression in pulmonary sarcoidosis.

The CC chemokine receptor 5 (CCR5) binds the chemokine ligands RANTES (CCL5) and MIP-1alpha (CCL3), which have been implicated in the development of alveolitis in sarcoidosis. We have, therefore, investigated CCR5 mRNA expression in bronchoalveolar lavage fluid (BALF) cells from patients with sarcoidosis. Further, we explored whether there was any association between CCR5 mRNA expression and the presence of the CCR5Delta32 DNA polymorphism. Semiquantitative RT-PCR was used to determine CCR5 mRNA expression from BALF cells from 16 control subjects (C) and 39 patients with sarcoidosis (S). The data on the CCR5Delta32 polymorphism, determined by PCR-SSP, were available for 37 patients. CCR5 mRNA expression was significantly upregulated in sarcoidosis (median+/-SEM, C, 0.00+/-0.07; S, 0.12+/-0.07; P<0.05). When patients were evaluated according to their CCR5Delta32 genotype, an interesting trend emerged with Delta32 positive patients (wt, mt) expressing less mRNA than the patients with both wild-type alleles (wt, wt): 0.00+/-0.09, and 0.26+/-0.09, respectively; P>0.05). In conclusion, upregulation of CCR5 mRNA in BALF of patients with sarcoidosis is consistent with its chemokine ligands RANTES and MIP-1alpha playing a pivotal role in inflammatory cell recruitment to disease sites. Though the data from this pilot study had no clinical correlations we suggest that further studies are warranted on the role of this Th1 subset marker in the pathogenesis of sarcoidosis.