Seroprevalence of herpes simplex virus type 1 and type 2 in Thuringia, Germany, 1999 to 2006.

The prevalence of herpes simplex virus (HSV) type-specific IgG was determined in sera taken in 1999 to 2006 from 1,100 children aged 0­18 years, 800 blood donors and 200 pregnant women in Thuringia, Germany, using tests based on the HSV glycoproteins (g) gG. By the age of 10­12 years, HSV-1 IgG prevalence reached 57.3%, rising to 69.3% by the age of 16­18 years and to 78.0% by the age of 28­30 years. Between 2.7% and 4.7% of the children aged up to 15 years had HSV-2 antibodies, increasing to 7.3% at the age of 16­18 years and to 13.6% among adults. The prevalence of HSV-1 antibodies among girls was significantly lower than among boys and a significantly higher prevalence of HSV-2 IgG in women than in men was detected. The reduced incidence of HSV-1 infections during childhood, especially in girls, has to be followed up since a higher number of primary HSV-2 infections may result. Between 2.7% and 4.7% of all children tested seemed to acquire HSV-2 by intrauterine or neonatal infection. We also compared the use of gG-1 with gC-1: the agreement of 97.2% between the two ELISAs suggests that gG-1 and gC-1 can be considered equivalent antigenic targets.

Identification and evaluation of a combination of chlamydial antigens to support the diagnosis of severe and invasive Chlamydia trachomatis infections.

Chlamydia trachomatis is the most common sexually transmitted organism in industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.

Western immunoblotting with five Treponema pallidum recombinant antigens for serologic diagnosis of syphilis.

Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species.

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of genes encoding outer surface protein C (OspC) of Borrelia burgdorferi sensu lato: relationship to ospA genotype and evidence of lateral gene exchange of ospC.

It has been shown by analysis with monoclonal and polyclonal antibodies that outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is highly heterogeneous. To determine if the heterogeneity has a genetic basis, the genes of 18 different B. burgdorferi sensu lato strains have been amplified by PCR, cloned, and sequenced. The ospC genes could be amplified from all strains tested, even from two strains which did not express OspC in detectable amounts. Among the 18 strains, 16 significantly different types of ospC sequences have been found. The sequence identities of the deduced amino acid sequences of different ospC genotypes range between 62 and 80% (determined without the leader peptide). The sequences range between 62 and 80% (determined without the leader peptide). The sequences correspond to one of the 13 OspC types distinguishable by analysis with monoclonal antibodies (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Roessler, E. Soutschek, and R. C. Johnson, J. Clin. Microbiol. 33:103-109, 1995) or represent additional types. Two completely new types were found, and OspC type 8 (which was found in Borrelia afzelii and Borrelia garinii) could be divided into two groups with different sequences but the same antibody pattern. Thus, strains belonging to different species or OspA serotypes were always significantly different in their ospC sequences. This was also confirmed by ospA sequence analysis. Interestingly, some strains of the same OspA serotype or genotype were very heterogeneous with respect to OspC, while others had nearly identical OspC proteins. Such groups of strains were found among B. burgdorferi sensu stricto, B. afzelii, and B. garinii strains. Cluster analysis of 5'-terminal and 3'-terminal stretches of ospC suggested recent intragenic recombination events in the ospC gene at least one B. afzelii strain. In addition, other recombination events between ancestors of strains belonging to the same or different species were evidenced by this type of analysis.

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains.

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.

Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype.

Molecular analyses of the genes encoding OspC, a major immunodominant protein of Borrelia burgdorferi sensu lato, revealed a considerable degree of heterogeneity. In the present study, we investigated whether a similar heterogeneity of the OspC phenotype can be shown by analysis with monoclonal antibodies (MAbs). Thirteen OspC-specific MAbs (L22 MAbs) were produced by immunizing mice with either different combinations of whole-cell antigens or recombinantly expressed OspCs cloned from strains belonging to different Borrelia spp. Ten of them differed in their reactivities with various strains. Western blot (immunoblot) analyses of 38 B. burgdorferi sensu lato strains resulted in 13 different reactivity patterns. These 13 different patterns were observed among only six different OspA serotypes, indicating that OspC is more heterogeneous than OspA. Patterns 1 to 4 were present only in B. burgdorferi sensu stricto, patterns 5 to 7 were present only in Borrelia afzelii, and patterns 9 to 13 were present only in Borrelia garinii. Pattern 8 was observed among B. afzelii and B. garinii strains but not among B. burgdorferi sensu stricto strains. One L22 MAb (2B8) recognized a common OspC-specific epitope of all 38 B. burgdorferi sensu lato strains analyzed, and another one (22C11) recognized a common epitope of OspC from both B. afzelii and B. garinii and was not reactive with OspC from B. burgdorferi sensu stricto. Western blot and sequence analysis of truncated OspCs located the 22C11 epitope as well as a species-specific sequence motif between amino acids 20 and 35. Other broadly reactive L22 MAbs were 10D3, 1F8, and 7G5. Some L22 MAbs (1C3, 1C3, 12E5, 1B11, 1F10, and 6C8) bound to epitopes present only in a few strains. Relapsing fever borreliae (Borrelia hermsii, Borrelia turicatae, and Borrelia duttoni) were nonreactive, with the following exception: three L22 MAbs (2B8, 6C4, and 10C5) recognized an abundantly expressed 20-kDa-range protein of B. turicatae. Because OspC is an immunodominant protein during the early immune response in Lyme borreliosis and has been shown to be effective as a vaccine in an animal model, our findings have important implications for the development of diagnostic reagents as well as vaccine research.

Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato.

Immunodominant proteins are variable in molecular and antigenic structure among different genospecies of Borrelia burgdorferi sensu lato. We have recently developed an immunoblot using five recombinant antigens: the chromosomal-encoded B. burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof, and the plasmid-encoded outersurface proteins A (OspA) and C (OspC). In the present study the same antigens (derived from strain PKo, genospecies B. afzelii) were compared with the homologous recombinant proteins from strain B31 (genospecies B. burgdorferi sensu stricto) and with OspA, OspC and the internal flagellin fragment from strain PBi (genospecies B. garinii). Patients with neuroborreliosis (n = 28) and patients with acrodermatitis chronica atrophicans (n = 20) were investigated in the IgG immunoblot; the IgM immunoblot was performed only in patients with neuroborreliosis. There was a small increase in the detection rate of OspA-specific IgG or IgM antibodies using the different variants of recombinant OspA; however, OspA remained an insensitive antigen for antibody detection in Lyme borreliosis. The same was true to OspC-specific IgG antibodies. The sensitivity of OspC, which is the immunodominant antigen for IgM antibody detection, could not be increased using recombinant antigens derived from different strains. However, some sera which were negative in the recombinant immunoblot reacted with OspC in the conventional immunoblot using B. burgdorferi whole cell lysate as antigen. The most unexpected finding was the high degree of immunological heterogeneity of the internal flagellin fragments: IgG antibodies were detected in 18 of 48 patients using B31 fragments, in 25 of 48 using PKo fragments, in 23 of 48 using PBi fragments versus 33 of 48 when the three recombinant proteins were combined. PKo-derived fragments were more sensitive for antibody detection in patients with acrodermatitis chronica atrophicans, B31- and PBi-derived fragments for antibody detection in patients with neuroborreliosis. This is in agreement with the fact that isolates from patients with neuroborreliosis are predominantly belonging to the genospecies B. burgdorferi sensu stricto and B. garinii. For detection of IgM antibodies in sera from patients with neuroborreliosis, recombinant internal fragments derived from strains B31 and PBi were more sensitive than the PKo-derived fragment. The best discrimination between neuroborreliosis sera and control sera was achieved when the IgM blot was performed using recombinant internal flagellin fragments derived from strains PKo and PBi and OspC derived from B31 or PKo.

Molecular characterization of the p100 gene of Borrelia burgdorferi strain PKo.

The p100 gene coding for the p100 protein of Borrelia burgdorferi strain PKo has been cloned, sequenced and expressed in Escherichia coli. An open reading frame including upstream and downstream sequences with potential translation and transcription signals could be identified. The reading frame consists of 1989 nucleotides corresponding to a protein of 663 amino acids and a calculated molecular mass of 75.8 kDa. The protein has a leader peptide and is processed without modification at the N-terminus. A high percentage of amino acid sequence identity could be found to the high-molecular mass protein p83/p93 of B. burgdorferi strain B31.

Recombinant immunoblot in the serodiagnosis of Lyme borreliosis. Comparison with indirect immunofluorescence and enzyme-linked immunosorbent assay.

A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encoded Borrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans, n = 31; neuroborreliosis state II, n = 60; Lyme arthritis, n = 24 and acrodermatitis chronica atrophicans, n = 19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.

Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi.

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.

Genetic heterogenity of the genes coding for the outer surface protein C (OspC) and the flagellin of Borrelia burgdorferi.

The ospC gene coding for the outer surface protein OspC and the fla gene coding for the flagellin have been investigated in three different Borrelia burgdorferi sensu lato strains. These strains (the North American strain B31 and the European strains PKo and PBi) derive from various biological sources (lxodes dammini, human skin and human CSF) and belong to three different B. burgdorferi OspA serotypes and genospecies (OspA serotype 1, B. burgdorferi sensu stricto; OspA serotype 2, group VS461 and OspA serotype 4, B. garinii, respectively). The ospC and fla genes of the respective strains have been amplified by polymerase chain reaction, cloned in pUC8 and sequenced. The fla as well as the ospC genes were different among the three strains investigated. In general the fla genes are more conserved than the ospC genes. The fla genes have the same length of 1008 nucleotides coding for proteins of 336 amino acids, whereas the ospC genes differ in length. The ospC genes of strains B31, PKo and PBi have 630, 636 and 621 nucleotides encoding proteins of 210, 212 and 207 amino acids, respectively. The ospC genes exhibit sequence identities between 70% and 74% among each other, sequence identities of the fla genes are in the range 96-97%. The ospC genes could be expressed in Escherichia coli to obtain proteins with and without leader peptides. The expression of the fla gene and an internal gene fragment resulted in the complete flagellin protein and a truncated protein (amino acids 129-251). The different ospC and fla gene products were immunoreactive with monoclonal antibodies and human sera and, thus, enlarge the spectrum of recombinant antigens to improve antibody detection in patients with Lyme borreliosis.

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.

Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection.

Serious infection due to Borrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains of B. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived from B. burgdorferi strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the same B. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine against B. burgdorferi.

Antigenic variation and strain heterogeneity in Borrelia spp.

Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i.e. B. burgdorferi and the relapsing fever borreliae. In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i.e. from inner sites to telomeric sites of the plasmid). As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon. In B. burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease. Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC). There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon. With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B. burgdorferi is immunologically and genetically more heterogeneous, as previously believed. The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level. The same degree of genetic non-identity was observed for the pC protein. Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity. Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E. coli and these proteins are immunologically reactive. This allows further research for development of vaccines and diagnostic tools. B. burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors. Comparison of the different methods has shown good agreement when the same strains have been investigated. No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice. A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed. Serotyping a large number of B. burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa protein (pC) in Escherichia coli.

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5' end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.

Genetic polymorphism of the gene encoding the outer surface protein A (OspA) of Borrelia burgdorferi.

The genes coding for the outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme borreliosis have been cloned and sequenced. Two German strains (skin isolate PKo and cerebrospinal fluid isolate PBi) have been analyzed. Using an OspA-specific monoclonal antibody (L32 2E7) for immunological screening of a genomic pUC18 library of B. burgdorferi strain PKo, and OspA-producing clone was detected and subclones containing the open reading frame were constructed. The gene coding for the OspA protein of B. burgdorferi strain PBi was amplified using polymerase chain reaction (PCR) and cloned in pUC8. The open reading frame of both ospA genes consists of 822 nucleotides corresponding to a protein of 273 amino acids. Both proteins have a calculated molecular mass of 29.6 kDa. Molecular analysis revealed significant differences between each other and to already-published sequences of ospA of B. burgdorferi strains B31, ZS7 and N40 (the ospA genes of B31, ZS7 and N40 are nearly identical). The deduced amino acid sequences of the OspA protein of strains PKo and PBi showed a homology of 83% to each other and 77% and 80%, respectively, to OspA protein of strain B31. The three proteins contain a variable middle region, whereas the N and the C terminus are conserved. This unexpected high dissimilarity of the ospA genes may be important in respect to vaccination studies and diagnostic procedures (i.e., development of PCR primers or serodiagnostic antigens). Moreover, the molecular heterogeneity of OspA confirms three out of seven immunologically defined OspA serotypes of a recently proposed OspA serotyping system.

Immunodominant proteins of Borrelia burgdorferi, the etiological agent of lyme borreliosis.

Lyme borreliosis, a multisystem disorder involving the skin, the nervous system, the heart, the joints and many other organs, is a worldwide infectious disease which is transmitted by ticks of the Ixodes complex. Most frequently diagnosis is accomplished by detection of antibodies because the Borrelia are difficult to cultivate. Present serodiagnostic methods, however, are impaired by low sensitivity and unspecific reactions. The selection of immunodominant antigens with low cross-reactivity to other bacteria should improve antibody detection. Borrelia burgdorferi proteins have been analysed for cross-reactivity with immune sera from unrelated bacteria, and sera from patients with different stages of the disease. Suitable antigens for improving serodiagnosis have been detected and are reported here. In view of the immunological heterogeneity of Borrelia proteins, sensitivity of antibody detection may possibly be increased by using recombinant antigens derived from different strains. Immunization with recombinant OspA (a flagellum-associated protein) from a North American isolate protected mice from the challenge with three North American isolates. However, for development of an effective vaccine (especially in Europe), the heterogeneity of OspA has to be considered.

Purification of a recombinantly produced transmembrane protein (gp41) of HIV I.

The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies. The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli. The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell. Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures. Growth conditions of the recombinant E. coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen. Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components. For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used. After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed. The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important. Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.