Ariadna Sadziene

A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization.

A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferis invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.

Expression of the flagellin gene in Borrelia is controlled by an alternative sigma factor

The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level. The flagellin genes of two relapsing fever Borrelia species, B. hermsii and B. crocidurae, three Lyme disease genomic species, B. burgdorferi, B. afzelii and B. garinii, and the avian borreliosis agent, B. anserina, were compared and showed an 85-93% sequence identity to each other. Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical. Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B. hermsii (as transcribed in Escherichia coli) and B. burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes. The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B. hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria. Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.

Experimental immunization against Lyme borreliosis with recombinant Osp proteins: An overview

SummaryInterest in human and veterinary vaccines against Lyme borreliosis is growing. Both whole cell immunization and subunit vaccines can protect against infection withBorrelia burgdorferi. For development of a human vaccine the focus has been on a subunit vaccine. The most promising candidate is OspA, a major outer membrane lipoprotein ofB. burgdorferi sensu lato. Of Osp proteins A through D, OspA shows the least variability between strains in its sequence and in the level of its expression. Borreliae in ticks express OspA. Antibodies to OspA kill borreliaein vitro and provide passive protection in mice. Active immunization of mice with OspA provides protection against challenge by syringe inoculation or tick bite. The lipid moiety of the OspA is necessary for immunogenicity in the absence of a potent adjuvant. A recombinant OspA-based vaccine is already in clinical trials. Although there is compelling evidence that immunization with OspA will provide protection, questions remain regarding the duration of protection from such immunization, the necessity to have a minimum level of neutralizing antibodies at all times for protection, and the relationship of an immune response to OspA and autoimmune features of Lyme borreliosis. The experimental aspects of immunization with Osp-A based constructs and other Lyme vaccine candidates are reviewed and discussed.ZusammenfassungDie Entwicklung humaner und veterinärmedizinischer Impfstoffe gegen die Lyme Borreliose gewinnt zunehmend an Interesse. Ein Infektionsschutz gegenBorrelia burgdorferi kann sowohl durch eine Ganzzell- wie durch Subunit-Vakzinen erreicht werden. Bei der Entwicklung einer humanen Vakzine wurde das Hauptgewicht auf Subunit-Vakzine gelegt. Am vielversprechendsten ist OspA, ein wichtiges Lipoprotein der äußeren Membran vonB. burgdorferi sensu lato. OspA weist in seiner Aminosäuresequenz und dem Expressionsgrad unter der Reihe von Osp-Proteinen von A–D die geringste Stamm-Variabilität auf. In Zecken lebende Borrelien exprimieren OspA. Antikörper gegen OspA töten Borrelienin vitro ab und vermitteln bei Mäusen einen passiven Schutz. Die aktive Immunisierung von Mäusen mit OspA bietet gegen die Inokulation durch Injektion oder durch Zeckenstich einen Schutz. Die Lipid-Verbindung von OspA ist für deren Immunogenität erforderlich, wenn nicht ein potentes Adjuvans eingesetzt wird. Eine rekombinante OspA-Vakzine befindet sich bereits in klinischer Prüfung. Obwohl schlüssige Beweise dafür vorliegen, daß die Immunisierung mit OspA protektiv ist, bleiben Fragen zur Dauer des Schutzes durch eine solche Immunisierung, die Notwendigkeit eines anhaltenden Minimalspiegels neutralisierender Antikörper für den Schutz und die Beziehung zwischen Immunantwort gegen OspA und die Autoimmun-Komponente der Lyme Borreliose zu klären. Die experimentellen Aspekte der Immunisierung mit Vakzine-Konstrukten auf der Basis von OspA und anderen Kandidaten von Lyme Impfstoffen werden in einer Übersicht dargestellt und diskutiert.Interest in human and veterinary vaccines against Lyme borreliosis is growing. Both whole cell immunization and subunit vaccines can protect against infection withBorrelia burgdorferi. For development of a human vaccine the focus has been on a subunit vaccine. The most promising candidate is OspA, a major outer membrane lipoprotein ofB. burgdorferi sensu lato. Of Osp proteins A through D, OspA shows the least variability between strains in its sequence and in the level of its expression. Borreliae in ticks express OspA. Antibodies to OspA kill borreliaein vitro and provide passive protection in mice. Active immunization of mice with OspA provides protection against challenge by syringe inoculation or tick bite. The lipid moiety of the OspA is necessary for immunogenicity in the absence of a potent adjuvant. A recombinant OspA-based vaccine is already in clinical trials. Although there is compelling evidence that immunization with OspA will provide protection, questions remain regarding the duration of protection from such immunization, the necessity to have a minimum level of neutralizing antibodies at all times for protection, and the relationship of an immune response to OspA and autoimmune features of Lyme borreliosis. The experimental aspects of immunization with Osp-A based constructs and other Lyme vaccine candidates are reviewed and discussed. Die Entwicklung humaner und veterinärmedizinischer Impfstoffe gegen die Lyme Borreliose gewinnt zunehmend an Interesse. Ein Infektionsschutz gegenBorrelia burgdorferi kann sowohl durch eine Ganzzell- wie durch Subunit-Vakzinen erreicht werden. Bei der Entwicklung einer humanen Vakzine wurde das Hauptgewicht auf Subunit-Vakzine gelegt. Am vielversprechendsten ist OspA, ein wichtiges Lipoprotein der äußeren Membran vonB. burgdorferi sensu lato. OspA weist in seiner Aminosäuresequenz und dem Expressionsgrad unter der Reihe von Osp-Proteinen von A–D die geringste Stamm-Variabilität auf. In Zecken lebende Borrelien exprimieren OspA. Antikörper gegen OspA töten Borrelienin vitro ab und vermitteln bei Mäusen einen passiven Schutz. Die aktive Immunisierung von Mäusen mit OspA bietet gegen die Inokulation durch Injektion oder durch Zeckenstich einen Schutz. Die Lipid-Verbindung von OspA ist für deren Immunogenität erforderlich, wenn nicht ein potentes Adjuvans eingesetzt wird. Eine rekombinante OspA-Vakzine befindet sich bereits in klinischer Prüfung. Obwohl schlüssige Beweise dafür vorliegen, daß die Immunisierung mit OspA protektiv ist, bleiben Fragen zur Dauer des Schutzes durch eine solche Immunisierung, die Notwendigkeit eines anhaltenden Minimalspiegels neutralisierender Antikörper für den Schutz und die Beziehung zwischen Immunantwort gegen OspA und die Autoimmun-Komponente der Lyme Borreliose zu klären. Die experimentellen Aspekte der Immunisierung mit Vakzine-Konstrukten auf der Basis von OspA und anderen Kandidaten von Lyme Impfstoffen werden in einer Übersicht dargestellt und diskutiert.

Lyme Borreliosis in Lithuania

The first cases of Lyme borreliosis in Lithuania were diagnosed in 1987, since when the number of reported cases has varied between 50 and 300 per year. In 1988, 354 cases were found through active surveillance. During 1988-91, 3,820 Ixodes ricinus ticks were collected in Lithuania; 11% of adult and 1% of nymphal ticks were infected with borrelia as determined by dark-field microscopy. Infected ticks were found in 27/31 (87%) of the regions investigated. Two isolates of Borrelia were obtained by in vitro cultivation and were shown to be Borrelia burgdorferi according to their reactions with species-specific monoclonal antibodies. From a survey of 6,187 people we estimated that 2,811/100,000 of the Lithuanian population are bitten by ticks every year. The prevalence of antibodies to B. burgdorferi measured by indirect IFA in healthy people varied between 4% and 32%, depending upon occupation and exposure to ticks.