Selective binding of Borrelia burgdorferi OspE paralogs to factor H and serum proteins from diverse animals: possible expansion of the role of OspE in Lyme disease pathogenesis

The binding of Borrelia burgdorferi OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.     

Changes in temporal and spatial patterns of outer surface lipoprotein expression generate population heterogeneity and antigenic diversity in the Lyme disease spirochete,Borrelia burgdorferi

Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi.     

OspE-related,OspF-related,and Elp lipoproteins are immunogenic in baboons experimentally infected with Borrelia burgdorferi and in human lyme disease patients

Presently, the rhesus macaque is the only nonhuman primate animal model utilized for the study of Lyme disease. While this animal model closely mimics human disease, rhesus macaques can harbor the herpes B virus, which is often lethal to humans; macaques also do not express the full complement of immunoglobulin G (IgG) subclasses found in humans. Conversely, baboons contain the full complement of IgG subclasses and do not harbor the herpes B virus. For these reasons, baboons have been increasingly utilized as the basis for models of infectious diseases and studies assessing the safety and immunogenicity of new vaccines. Here we analyzed the capability of baboons to become infected with Borrelia burgdorferi, the agent of Lyme disease. Combined culture and PCR analyses of tick- and syringe-infected animals indicated that baboons are a sufficient host for B. burgdorferi. Analysis of the antibody responses in infected baboons over a 48-week period revealed that antibodies are generated early during infection against many borrelial antigens, including the various OspE, OspF, and Elp paralogs that are encoded on the ubiquitous 32-kb circular plasmids (cp32s). By using the baboon sera generated by experimental infection it was determined that a combination of two cp32-encoded lipoproteins, OspE and ElpB1, resulted in highly specific and sensitive detection of B. burgdorferi infection. An expanded analysis, which included 39 different human Lyme disease patients, revealed that a combination of the OspE and ElpB1 lipoproteins could be the basis for a new serodiagnostic assay for Lyme disease. Importantly, this novel serodiagnostic test would be useful independent of prior OspA vaccination status.     

Comprehensive analysis of the factor h binding capabilities of borrelia species associated with lyme disease: delineation of two distinct classes of factor h binding proteins

Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing. Here we demonstrate significant differences in the fH binding capabilities of species of the Borrelia burgdorferi sensu lato complex. The percentages of B. burgdorferi, B. afzelii, and B. garinii bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively. The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes. Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the B. burgdorferi sensu lato complex.     

Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection

The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that the ospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.     

Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice.

We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.     

Lysine-Dependent Binding of OspE to the C-terminus of Factor H Mediates Complement Resistance in Borrelia burgdorferi

Serum resistance of Borrelia burgdorferi strains belonging to the B. afzelii and B. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor H. We recently reported that factor H binding by B. burgdorferi is due to inducible expression of several approximately 20 kDa plasmid‐encoded, surface‐exposed lipoproteins related to OspE (e.g. ErpA, ErpP and P21). In addition, a second class of factor H‐binding proteins of approximately 27–35 kDa has been described. The OspE‐related lipoproteins are dramatically induced by B. burgdorferi during transmission from its tick vector into the mammalian host. The induction of OspE‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. The goal of the present study was to define the factor H‐binding regions of OspE‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (Biacore). The combined studies revealed that the C‐terminal regions of both human and mouse factor H (SCRs 18–20) specifically bind to OspE‐related lipoproteins. We also found FHR‐1, whose C‐terminal SCRs 3–5 are homologous to SCRs 18–20 of factor H, to bind to OspE. Peptide mapping revealed five putative regions (designated I‐V) in OspE that could directly interact with factor H. Deleting the C‐terminal 15 amino acid residues from region V of P21 abolished its ability to bind factor H. At the same time, however, synthetic peptides corresponding to the C‐termini of OspE, P21 and ErpP did not inhibit factor H binding to OspE. Thus, the C‐terminal‐binding region V appears to be necessary but not sufficient for factor H binding. When a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor H‐binding regions were mutated to alanines, we observed that lysines in the factor H‐binding regions of OspE were required for factor H binding. The combined data have revealed that key lysine residues in OspE‐related lipoproteins and ionic interactions are crucial for factor H interactions. Furthermore, binding of OspE to the C‐termini of both mouse and human factor H suggests that Borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. In Borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the OspE sequences as well as in the expression of factor H‐binding proteins may account for their susceptibility to serum lysis.     

The OspE-related proteins inhibit complement deposition and enhance serum resistance of Borrelia burgdorferi, the lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction.     

Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi

The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in Borrelia burgdorferi. To examine the extent to which the 10 members of the Mlp family in B. burgdorferi strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when B. burgdorferi replicated under various cultivation conditions. All of the Mlps were upregulated coordinately when B. burgdorferi was cultivated at either elevated temperature, reduced culture pH, or increased spirochete cell density. Inasmuch as the expression of OspC is influenced by a novel RpoN-RpoS regulatory pathway, similar induction patterns for OspC and the Mlp paralogs prompted an assessment of whether the RpoN-RpoS pathway also was involved in Mlp expression. In contrast to wild-type B. burgdorferi, both RpoN- and RpoS-deficient mutants were unable to upregulate OspC or the Mlp paralogs when grown at lower pH (6.8), indicating that pH-mediated regulation of OspC and Mlp paralogs is dependent on the RpoN-RpoS pathway. However, when RpoN- or RpoS-deficient mutants were shifted from 23 degrees C to 37 degrees C or were cultivated to higher spirochete densities, some induction of the Mlps still occurred, whereas OspC expression was abolished. The combined findings suggest that the Mlp paralogs are coordinately regulated as a family and have an expression profile similar, but not identical, to that of OspC. Although Mlp expression as a family is influenced by the RpoN-RpoS regulatory pathway, there exists at least one additional layer of gene regulation, yet to be elucidated, contributing to Mlp expression in B. burgdorferi.     

Environmental regulation and differential production of members of the Bdr protein family of Borrelia burgdorferi

Borrelia burgdorferi B31MI carries 18 plasmid-carried genes that form the bdr gene family. The bdr genes of B. burgdorferi encode proteins that form three distinct subfamilies, the BdrD, BdrE, and BdrF subfamilies. bdr orthologs have been demonstrated to be carried by all Borrelia species analyzed, and their widespread distribution suggests that they play an important genus-wide functional role. The biological rationale for maintaining 18 bdr alleles has not been defined. It is our hypothesis that specific paralogs function in different environments and are differentially expressed in response to environmental conditions. As a first step in testing this hypothesis, the production patterns of the Bdr proteins in spirochetes grown under a variety of conditions were assessed through immunoblot analyses. The influence of temperature, serum deprivation, tick feeding, and the mammalian environment on Bdr production was evaluated. These analyses revealed that the synthesis of some Bdr paralogs is environmentally regulated. The production of BdrF(2,) BdrF(1), BdrE(4), and BdrE(5) were upregulated in host-adapted bacteria, while the production levels of other Bdr paralogs were influenced by temperature and serum starvation. These observations suggest that different Bdr paralogs function in different biological environments and provide insight into the biological basis for maintaining multiple members of this gene family.     

Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis

The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.     

The idiotypic network in antinuclear-antibody-associated diseases

Antinuclear antibodies (ANA) entail a large group of autoantibodies (Abs) that bind certain nuclear antigens. The ANA test is a useful screening test for many autoimmune diseases and the presence of a specific binding pattern directs secondary testing for specific Abs associated with the suspected disease. Idiotypes (Ids) are the antigenic constitution of the variable region of an Ab, and they are recognized by anti-Ids Abs. The Id network is composed of interacting Abs in which the Id determinants of each Ab are complemented by those of another. It has a role in both physiologic and pathologic conditions. In this communication, we review the induction of autoimmune diseases via Id manipulation, the Ids of some ANA-associated Abs (DNA, SS-A, SS- B, Sm Abs), the pathogenic role of Abs carrying Ids, and the clinical implications of the Id network in autoimmunity.     

Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.     

Immunoblotting with monoclonal antibodies: loss of immunoreactivity with human immunoglobulins arises from polypeptide chain separation

Immunoblotting has been used to study the antigen binding characteristics of 5 monoclonal antibodies (Mc/Abs) against human Ig (1 anti-kappa, 2 anti-gamma and 2 anti-delta chain. Of the 4 Mc/Abs only 1 (the anti-kappa chain Mc/Ab) reacted with its antigen when blotted from reducing SDS polyacrylamide gels. However, the 4 Mc/Abs which recognise immunoglobulin heavy chains were able to bind their antigens when blotted from native or non-reducing SDS gels. The lack of reactivity of the latter Mc/Abs in blots from reduced SDS gels may be attributed to the separation of Ig which occurs during electrophoresis after the -S-S- bonds are broken. It may be concluded that the conformation of Ig heavy chains is considerably altered when Ig molecules are disrupted and Ig chains separated, and several heavy chain determinants are lost during this process. Therefore determinants recognised by the anti-heavy chain Mc/Abs are most likely to be of the 'conformational' type whereas the anti-light chain Mc/Ab may well recognise a purely sequential determinant.     

Major histocompatibility complex class II-independent generation of neutralizing antibodies against T-cell-dependent Borrelia burgdorferi antigens presented by dendritic cells: regulation by NK and gammadelta T cells

We previously showed that adoptive transfer of Borrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized with B. burgdorferi-pulsed MHC class II-deficient (MHC class II(-/-)) DCs mounted a humoral response against protective antigens, including B. burgdorferi outer surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by gammadelta T cells. In contrast, anti-OspA Ab production required the presence of alphabeta T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II(-/-) DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as gammadelta and NK cells, can influence the generation of a protective humoral response against B. burgdorferi antigens.     

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.     

Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC.

Outbred mice were immunized with the soluble fraction of a crude Escherichia coli lysate containing either recombinant outer surface protein C (OspC or P39 of Borrelia burgdorferi B31 (low passage). Following seroconversion, the mice were challenged with an infectious dose of B. burgdorferi B31 via the natural transmission mode of tick bite. Three mice immunized with P39 were not protected; however, all 12 of the recombinant OspC-immunized mice were protected from infection as assayed by culture and serology. Although OspC has been shown to be a protective immunogen against challenge with in vitro-cultured borrelia administered by needle, this study is the first to demonstrate OspC effectiveness against tick-borne spirochetes. Following feeding, all ticks still harbored B. burgdorferi, suggesting that the mechanism of protection is not linked to destruction of the infectious spirochete within the tick. In a separate experiment, groups of four mice were immunized with protein fractions from B. burgdorferi B31 purified by preparative gel electrophoresis in an attempt to identify potential protective antigens. Many of these mice developed high-titer-antibody responses against OspC, but curiously the mice were susceptible to B. burgdorferi infection via tick bite. These results suggest that the protective epitope(s) on OspC is heat sensitive/conformational, a finding which has implications in vaccine development.     

C-terminal invariable domain of VlsE may not serve as target for protective immune response against Borrelia burgdorferi

VlsE, the variable surface antigen of the Lyme disease spirochete, Borrelia burgdorferi, contains two invariable domains, at the amino and carboxyl termini, respectively, which collectively account for approximately one-half of the entire molecule's length and remain unchanged during antigenic variation. It is not known if these two invariable domains are exposed at the surface of either the antigen or the spirochete. If they are exposed at the spirochete's surface, they may elicit a protective immune response against B. burgdorferi and serve as vaccine candidates. In this study, a 51-mer synthetic peptide that reproduced the entire sequence of the C-terminal invariable domain of VlsE was conjugated to the carrier keyhole limpet hemocyanin and used to immunize mice. Generated mouse antibody was able to immunoprecipitate native VlsE extracted from cultured B. burgdorferi B31 spirochetes, indicating that the C-terminal invariable domain was exposed at the antigen's surface. However, this domain was inaccessible to antibody binding at the surface of cultured intact spirochetes, as demonstrated by both an immunofluorescence experiment and an in vitro killing assay. Mouse antibody to the C-terminal invariable domain was not able to confer protection against B. burgdorferi infection, indicating that this domain was unlikely exposed at the spirochete's surface in vivo. We concluded that the C-terminal invariable domain was exposed at the antigen's surface but not at the surface of either cultured or in vivo spirochetes and thus cannot elicit protection against B. burgdorferi infection.     

Immune evasion of Borrelia burgdorferi: mapping of a complement-inhibitor factor H-binding site of BbCRASP-3, a novel member of the Erp protein family

The causative agents of Lyme disease, Borrelia burgdorferi s.s., B. garinii, and B. afzelii, differ in their susceptibility to complement-mediated lysis. This phenomenon apparently depends on the expression of proteins termed complement regulator-acquiring surface proteins (CRASP) and their binding to the inhibitory plasma proteins factor H and FHL-1. To characterize these bacterial proteins in more detail we have now isolated from a B. burgdorferi expression library a novel factor H-binding protein. In accordance with our previous studies this protein was termed BbCRASP-3 and represents a novel member of the polymorphic Erp (OspE/F-related) protein family. On the basis of protease accessibility assays using intact spirochetes, BbCRASP-3 is identified as a surface-exposed protein and binds the C-terminal short consensus repeats of factor H. Applying deletion mutants of BbCRASP-3, the factor H-binding site was mapped to the nine-amino-acid motif LEVLKKNLK localized at the C-terminal end of BbCRASP-3. Factor H bound to BbCRASP-3 maintains its cofactor activity in factor I-mediated C3b inactivation. Binding of BbCRASP-3 to factor H can be inhibited by heparin, a physiological ligand of the complement regulator factor H. Blocking of factor-H-binding by soluble BbCRASP-3 leads to an increase of complement deposition on intermediate serum-resistant strain ZS7. In conclusion, BbCRASP-3 has been identified as a novel factor H-binding protein on B. burgdorferi which by conferring complement resistance to the pathogen may contribute to its persistence in the mammalian host.     

Molecular basis for immunoglobulin M specificity to epitopes in Cryptococcus neoformans polysaccharide that elicit protective and nonprotective antibodies

The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V(H)) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V(H) binding cavity to the corresponding residues in the 13F1 V(H) altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.