The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP ...types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).
An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with ...the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.
A New Approach to a Lyme Disease Vaccine Livey, Ian; O'Rourke, Maria; Traweger, Andreas ...
Clinical infectious diseases,
02/2011, Letnik:
52, Številka:
suppl_3
Journal Article
Recenzirano
Odprti dostop
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.
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-γ 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.
Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The ...sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.
Twelve selected strains of Bordetella pertussis were compared quantitatively for their ability to produce heat-labile toxin (HLT); all proved to be active producers, with only a three-fold range ...between the highest and the lowest. Bordet-Gengou agar, charcoal agar, modified Hornibrook medium and Stainer and Schölte (12G) medium differed little in their ability to support toxin production by three B. pertussis strains. However, cells grown on the solid media for 24 h were slightly more toxic than their counterparts grown for 72 h whereas in the liquid media the opposite was true. The concentration of iron in the medium did not influence HLT production, but high concentrations of nicotinic acid significantly reduced the HLT content of the cells. Crude preparations of toxin underwent only a 10% loss of toxicity per annum at -20 degrees C and were stable for up to 2 weeks at 4 degrees C. At 37 degrees C, toxicity was lost within a few days. The toxin was partially purified by a series of mild procedures and had a mol. wt by gel filtration of 89 000 +/- 10%. HLT was toxoided by treatment with formaldehyde to give a product which was immunogenic in rabbits but not in mice. Because anti-HLT could be absorbed out of the rabbit antisera by treatment with intact B. pertussis, it was concluded that some of the HLT in the bacteria is surface-exposed even though the main part may have a cytoplasmic location.
IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481v1 is located within 3 kbp of the start of the adenylate cyclase gene whilst IS481v2 ...is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481v1 or IS481v2.
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.
The present study outlines the characterization of a DNA-based immune response against the OspC antigen, one of the most promising candidates for a
Borrelia vaccine. Balb/c mice were injected ...intradermally with plasmid DNA encoding the OspC gene (lacking the natural leader sequence) under transcriptional control of the cytomegalovirus (CMV) promotor. Immunization with this construct elicited only a marginal response, which was drastically improved by a fusion construct containing the human tissue plasminogen activator (hTPA) signal sequence. The results indicate that for DNA-based immunization against OspC an ER-targeting signal may be necessary for both antibody production as well as cellular immune responses.
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