Enzyme-linked immunosorbent assay (ELISA) for detection of IgY Anti-Borrelia anserina antibodies in Gallus gallus domesticus / Ensaio de imunoadsorção enzimático (ELISA) indireto para detecção de anticorpos IgY Anti-Borreliaanserina em Gallus gallus domesticus

This study aimed to promote the standardization of an indirect, enzyme-linked immunosorbent assay (ELISA) for the serological detection of B. anserina in Gallus gallusdomesticus. An aliquoted sera from vaccinated chicken with B. anserina antigen (GI), experimental infected chickens with B. anserina (GII) and rustic poultry rearing of G. gallus (GIII) were tested with in-house ELISA developed to detect serum antibodies against B. anserina in G. gallus domesticus. On average, the experimentally infected chickens became positive at 9 DPI a mean ± standard deviation (SD) ODI value of 163.11 ± 70.65. The highest observed Optical Density Index (ODI) was 372.54 ± 132.39, at 26 DPI, and the highest overall ODI value was 626.51. The vaccinated chickens became positive between 8 and 10 DPV, with an ODI of 245.59 at 10 DPV, with an overall maximum ODI of 543.13. A total of 108 blood samples were collected from poultry raised on rustic farms. Of the total samples collected, 58.33% (63/108) were considered positive for B. anserina. The maximum ODI found among these rustic chickens was 283.24. This stardardization provided a sensitivity and specificity of 100%.

Este estudo teve como objetivo promover a padronização de um ensaio imunoenzimático indireto (ELISA) para a detecção sorológica de Borrelia anserina em Gallus gallus domesticus. Um frango vacinado com antígeno de B. anserina (GI), frangos infectados experimentalmente com B. anserina (GII) e frangos criados de forma rústica (GIII) foram testados com ELISA indireto in house desenvolvido para a detecção sorológica contra B. anserina em G. gallus domesticus. Em média, os frangos infectados experimentalmente tornaram-se positivos aos 9º dia pós-inoculação (DPI), um valor do índice de densidade óptica (ODI) médio ± desvio padrão (SD) de 163,11 ± 70,65. O maior ODI observado foi 372,54 ± 132,39, em 26ºDPI, e o maior valor geral de ODI foi 626,51. Os frangos vacinados tornaram-se positivos entre 8º e 10° DPV, com um ODI de 245,59 a 10 DPV, com um ODI máximo geral de 543,13. Um total de 108 amostras de sangue foram coletadas de aves criadas em fazendas rústicas. Do total de amostras coletadas, 58,33% (63/108) foram consideradas positivas para B. anserina. O ODI máximo encontrado entre essas galinhas rústicas foi 283,24. Essa padronização proporcionou sensibilidade e especificidade de 100%.

Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men.

OBJECTIVES: Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. METHODS: We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. RESULTS: In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. CONCLUSIONS: We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

Differences in clinicopathologic variables between Borrelia C6 antigen seroreactive and Borrelia C6 seronegative glomerulopathy in dogs.

BACKGROUND: Rapidly progressive glomerulonephritis has been described in dogs that seroreact to Borrelia burgdorferi, but no studies have compared clinicopathologic differences in Lyme-seroreactive dogs with protein-losing nephropathy (PLN) versus dogs with Borrelia-seronegative PLN. HYPOTHESIS/OBJECTIVES: Dogs with Borrelia C6 antigen-seroreactive PLN have distinct clinicopathologic findings when compared to dogs with Borrelia seronegative PLN. ANIMALS: Forty dogs with PLN and Borrelia C6 antigen seroreactivity and 78 C6-seronegative temporally matched dogs with PLN. METHODS: Retrospective prevalence case-control study. Clinical information was retrieved from records of dogs examined at the University of California, Davis, Veterinary Medical Teaching Hospital. Histopathologic findings in renal tissue procured by biopsy or necropsy of dogs with PLN were reviewed. RESULTS: Retrievers and retriever mixes were overrepresented in seroreactive dogs (P < .001). Seroreactive dogs were more likely to have thrombocytopenia (P < .001), azotemia (P = .002), hyperphosphatemia (P < .001), anemia (P < .001), and neutrophilia (P = .003). Hematuria, glucosuria, and pyuria despite negative urine culture were more likely in seroreactive dogs (all P ≤ .002). Histopathologic findings were consistent with immune-complex glomerulonephritis in 16 of 16 case dogs and 7 of 23 control dogs (P = 006). Prevalence of polyarthritis was not different between groups (P = .17). CONCLUSIONS AND CLINICAL IMPORTANCE: C6 seroreactivity in dogs with PLN is associated with a clinicopathologically distinct syndrome when compared with other types of PLN. Early recognition of this syndrome has the potential to improve outcomes through specific aggressive and early treatment.

Immunological Responses to the Relapsing Fever Spirochete Borrelia turicatae in Infected Rhesus Macaques: Implications for Pathogenesis and Diagnosis.

The global public health impact of relapsing fever (RF) spirochetosis is significant, since the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B-cell responses and the role of immune responses in pathogenicity, we infected rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (interleukin-1ß and tumor necrosis factor alpha), which are associated with preterm abortion. Moreover, a global decline in peripheral B-cell populations was observed in all animals at 14 days postinfection. Serological responses were also evaluated to assess the antigenicity of three surface proteins: BipA, BrpA, and Bta112. Interestingly, a distinction was observed between antibodies generated in nonhuman primates and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis but also for diagnostic and vaccine antigen identification and testing.

Positive 2-Tiered Lyme Disease Serology is Uncommon in Asymptomatic Children Living in Endemic Areas of the United States.

Knowing the frequency of positive Lyme disease serology in children without signs of infection facilitates test interpretation. Of 315 asymptomatic children from Lyme disease endemic regions, 32 had positive or equivocal C6 enzyme-linked immunoassays, but only 5 had positive IgG or IgM supplemental immunoblots (1.6%; 95% confidence interval: 0.7%-3.7%).

Seroreactivity to the C6 Peptide in Borrelia miyamotoi Infections Occurring in the Northeastern United States.

Background: There are no US Food and Drug Administration (FDA)-approved diagnostic tests for Borrelia miyamotoi infection, an emerging tick-borne illness in the United States. The purpose of this study was to evaluate whether the FDA-approved C6 peptide enzyme-linked immunosorbent assay (ELISA) currently used to diagnose Lyme disease may potentially serve as a diagnostic test for B. miyamotoi infections. Methods: Serum specimens from 30 patients from the northeastern United States with B. miyamotoi infection established by a polymerase chain reaction assay of a blood specimen were tested using the C6 ELISA. To reduce confounding with Borrelia burgdorferi coinfection, 6 sera were excluded: 3 from patients with a positive Western immunoblot for antibodies to B. burgdorferi and 3 from patients for whom immunoblot testing had not been performed. Results: Twenty-two of 24 (91.7% [95% confidence interval, 73.0%-98.8%]) evaluable B. miyamotoi patients were C6 ELISA reactive, principally on a convalescent-phase serum specimen. C6 ELISA index values were often well above the positive cutoff value of 1.1, exceeding 4 in 11 of the 22 (50.0%) C6 ELISA-reactive patients. Conclusions: Although previously regarded as a highly specific test for Lyme disease, the C6 ELISA is also regularly reactive on convalescent-phase serum samples of patients from the northeastern United States with B. miyamotoi infection.

Humoral Immunity in Mice Transplanted with Hematopoietic Stem Cells Derived from Human Umbilical Cord Blood Recapitulates That of Human Infants.

Immunodeficient mice transplanted with human hematopoietic stem cells (HSCs) have been referred to as "Human Immune System" (HIS) mice and are a translational platform for studying human immune responses in vivo. Human HSC sources used in generating HIS mice include fetal liver (FL), umbilical cord blood (CB), and adult bone marrow (BM). Since HSCs from FL, CB, and BM are produced at various stages of human development, we tested whether mice transplanted with these three HSCs differ in their immune responses. We found that compared with CB HSCs or FL HSCs, adult BM HSCs reconstitute the immune system poorly. The resulting HIS mice do not mount an antibody response to Borrelia hermsii infection and as a consequence suffer persistently high levels of bacteremia. While both CB and FL HSCs yield comparable levels of immune reconstitution of HIS mice resulting in robust anti-B. hermsii immune responses, FL HSC-transplanted mice exhibited a discernable difference in their human B cell maturity as identified by an increased frequency of CD10+ immature B cells and relatively smaller lymphoid follicles compared with CB HSC-transplanted mice. Although CB HSC-transplanted mice generated robust antibody responses to B. hermsii and specific protein antigens of B. hermsii, they failed to respond to Salmonella typhi Vi polysaccharide, a classical T cell-independent antigen. This situation resembles that seen in human infants and young children. Therefore, CB HSC-transplanted mice may serve as a translation platform to explore approaches to overcome the impaired antipolysaccharide responses characteristic of human infants.

Segregation Lag in Polyploid Cells of the Pathogen Genus Borrelia: Implications for Antigenic Variation
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Relapsing fever agents like Borrelia hermsii undergo multiphasic antigenic variation that is attributable to spontaneous DNA non-reciprocal transpositions at a particular locus in the genome. This genetic switch results in a new protein being expressed on the cell surface, allowing cells with that phenotype to escape prevailing immunity. But the switch occurs in only one of several genomes in these spirochetes, and a newly-switched gene is effectively "recessive" until homozygosity is achieved. The longer that descendants of the switched cell expressed both old and new proteins, the longer this lineage risks neutralization by antibody to the old protein. We investigated the implications for antigenic variation of the phenotypic lag that polyploidy would confer on cells. We first experimentally determined the average genome copy number in daughter cells after division during mouse infection with B. hermsii strain HS1. We then applied discrete deterministic and stochastic simulations to predict outcomes when genomes were equably segregated either linearly, i.e. according to their position in one-dimensional arrays, or randomly partitioned, as for a sphere. Linear segregation replication provided for a lag in achievement of homozygosity that was significantly shorter than could be achieved under the random segregation condition. For cells with 16 genomes, this would be a 4-generation lag. A model incorporating the immune response and evolved matrices of switch rates indicated a greater fitness for polyploid over monoploid bacteria in terms of duration of infection.

Borrelia-primed and -infected mice deficient of interleukin-17 develop arthritis after neutralization of gamma-interferon.

The immune mechanisms responsible for development of Lyme arthritis are partially understood with interleukin-17 (IL-17) and gamma-interferon (IFN-γ) playing a generally accepted role. Elevated levels of IL-17 and/or IFN-γ have been reported in samples from human Lyme arthritis patients and experimental mice. In addition, IL-17 and IFN-γ have been implicated in the onset of arthritis in Borrelia-primed and -infected C57BL/6 mice. Recently, we showed that IL-17-deficient mice developed swelling and histopathological changes consistent with arthritis in the presence of high levels of IFN-γ. We hypothesized that neutralization of IFN-γ in IL-17-deficient mice would inhibit Borrelia-induced arthritis. Our results, however, showed that swelling of the hind paws and histopathological changes of arthritis did not differ between Borrelia-primed and -infected IL-17-deficient and wild-type mice with or without neutralization of IFN-γ. We also found higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 in the popliteal lymph node cells of Borrelia-primed and -infected IL-17-deficient mice after neutralization of IFN-γ. These results suggest that multiple cytokines interact in the development of Borrelia-induced arthritis.

Borrelia miyamotoi-Associated Neuroborreliosis in Immunocompromised Person.

Borrelia miyamotoi is a newly recognized human pathogen in the relapsing fever group of spirochetes. We investigated a case of B. miyamotoi infection of the central nervous system resembling B. burgdorferi-induced Lyme neuroborreliosis and determined that this emergent agent of central nervous system infection can be diagnosed with existing methods.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity.

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

Ixodes ricinus salivary serpin IRS-2 affects Th17 differentiation via inhibition of the interleukin-6/STAT-3 signaling pathway.

Th17 cells constitute a subset of CD4(+) T lymphocytes that play a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here, we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of the interleukin-6 (IL-6)/STAT-3 signaling pathway. Following activation, mature dendritic cells produce an array of cytokines, including the pleiotropic cytokine IL-6, which triggers the IL-6 signaling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. The results presented extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune responses.

Maladjusted host immune responses induce experimental cerebral malaria-like pathology in a murine Borrelia and Plasmodium co-infection model.

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1ß and TNF-α, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.

IrFC - An Ixodes ricinus injury-responsive molecule related to Limulus Factor C.

Limulus Clotting Factor C is a multi-domain serine protease that triggers horseshoe crab hemolymph clotting in the presence of trace amounts of bacterial lipopolysaccharides. Here we describe and functionally characterize an homologous molecule, designated as IrFC, from the hard tick Ixodes ricinus. Tick Factor C consists of an N-terminal cysteine-rich domain, four complement control protein (sushi) modules, an LCCL domain, a truncated C-lectin domain and a C-terminal trypsin-type domain. Developmental expression profiling by quantitative real-time PCR revealed that the irfc mRNA is expressed in all stages including eggs. In tissues dissected from adult I. ricinus females, the irfc mRNA is present mainly in tick hemocytes and accordingly, indirect immunofluorescence microscopy localized IrFC intracellularly, in tick hemocytes. Irfc mRNA levels were markedly increased upon injection of sterile saline, or different microbes, demonstrating that the irfc gene transcription occurs in response to injury. This indicates a possible role of IrFC in hemolymph clotting and/or wound healing, although these defense mechanisms have not been yet definitely demonstrated in ticks. RNAi silencing of irfc expression resulted in a significant reduction in phagocytic activity of tick hemocytes against the Gram-negative bacteria Chryseobacterium indologenes and Escherichia coli, but not against the yeast, Candida albicans. This result suggests that IrFC plays a role in the tick primordial complement system and as such possibly mediates transmission of tick-borne pathogens.

Efficient B cell responses to Borrelia hermsii infection depend on BAFF and BAFFR but not TACI.

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI(-/-) mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii-specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

Microbiologic findings in acute facial palsy in children.

OBJECTIVE: Microbiologic causes of facial palsy in children were investigated. STUDY DESIGN: Prospective clinical study. SETTING: Tertiary referral center. PATIENTS: Forty-six children aged 0 to 16 years with peripheral facial palsy. INTERVENTIONS: Paired serum samples and cerebrospinal fluid were tested to find indications of microbes associated with facial palsy. The microbes tested were herpes simplex virus 1 and 2, varicella-zoster virus, human herpesvirus-6, Mycoplasma pneumoniae, Borrelia burgdorferi, influenza A and B virus, picorna, cytomegalovirus, parainfluenza virus, respiratory syncytial virus, coxsackie B5 virus, adenovirus, and enterovirus, Chlamydia psittaci, and Toxoplasma gondii. Besides the routine tests in clinical practice, serum and cerebrospinal fluid samples were tested with a highly sensitive microarray assay for DNA of herpes simplex virus 1 and 2; human herpes virus 6A, 6B, and 7; Epstein-Barr virus, cytomegalovirus, and varicella zoster virus. RESULTS: Incidence for facial palsy was 8.6/100,000/children/year. Cause was highly plausible in 67% and probable in an additional 11% of cases. Borrelia burgdorferi caused facial palsy in 14 patients (30%), varicella zoster virus in 5 (11%) (one with concomitant adenovirus), influenza A in 3 (6%), herpes simplex virus 1 in 2 (4%) (one with concomitant enterovirus), otitis media in 2 (4%), and human herpesvirus 6 in 2 (4%). Mycoplasma pneumoniae, neurofibromatosis, and neonatal age facial palsy affected 1 child (2%) each. CONCLUSION: Microbiologic etiology association of pediatric facial palsy could frequently be confirmed. Borreliosis was the single most common cause; hence, cerebrospinal fluid sampling is recommended for all pediatric cases in endemic areas. Varicella zoster virus accounted for 11% of the cases, being the second most common factor.

Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components.

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae.

Acrodermatitis chronica atrophicans with pseudolymphomatous infiltrates.

In this study, we describe the clinicopathologic features of pseudolymphomatous infiltrates found within lesions of acrodermatitis chronica atrophicans (ACA). We studied 11 patients (10 females, 1 male, age range 60-88 years). The diagnosis of ACA in all cases was confirmed by clinicopathologic correlation and positive serology for Borrelia. Histopathologic examination revealed prominent, pseudolymphomatous inflammatory cell infiltrates in all cases, with 2 distinct patterns. Eight of 11 cases showed a band-like lymphocytic infiltrate, exocytosis of lymphocytes and a fibrotic papillary dermis, similar to features seen in mycosis fungoides. The other 3 cases showed dense, nodular-diffuse dermal infiltrates with many plasma cells and without germinal centers. The plasma cells expressed both kappa and lambda immunoglobulin light chains with a polyclonal pattern in all 3 cases. In conclusion, ACA may present with pseudolymphomatous infiltrates showing both a T-cell and, less frequently, a B-cell pattern. These lesions need to be distinguished from a cutaneous lymphoma. In the context of the knowledge of Borrelia-associated cutaneous lymphomas, follow-up seems advisable in these cases.

N-terminal disulfide-bridging of Borrelia outer surface protein C increases its diagnostic and vaccine potentials.

OspC is the main target for IgM in early-stage Lyme disease. As such it is employed as its native or recombinant form in routine immunoassays for the determination of Borrelia-specific antibodies. However, recombinant OspC has so far not shown the antigenicity of the native protein. The latter contains an intrinsic signal sequence and an adjacent cysteine residue, the attachment site of the lipid membrane anchor which has been discussed to have an adjuvant effect on the immune reaction. In expression experiments, we have found a recombinant variant, an OspC covalently homodimerized via an N-terminal disulfide bridge, that shows a remarkably enhanced antigenicity without lipid attachment. Three such OspCs derived from different Borrelia strains were subsequently expressed in E. coli and purified under non-reducing conditions. In non-reducing SDS-PAGE, OspC(Δ1-18) exhibited a 48-kDa band of dimeric OspC. When incubated with IgM-OspC-positive human sera, the reaction at 48kDa was always stronger than at 24kDa of monomeric OspC(Δ1-18, C19G). A lineblot with OspC(Δ1-18) also showed a higher diagnostic accuracy than that obtained with OspC(Δ1-18, C19G) based on a higher affinity of IgM for the dimeric form. When used for the immunization of mice, dimeric OspC(Δ1-18) induced consistent high-titre antibodies against OspC, whereas OspC(Δ1-18, C19G) failed to provoke significant titres in some animals. We conclude that the disulfide-bridging of 2 OspC molecules via their N termini forms a complex that is more suitable for the determination of IgM-OspC and is a promising candidate for a monovalent vaccine.

B1b lymphocyte-derived antibodies control Borrelia hermsii independent of Fcα/µ receptor and in the absence of host cell contact.

The critical role of IgM in controlling pathogen burden has been demonstrated in a variety of infection models. In the murine model of Borrelia hermsii infection, IgM is necessary and sufficient for the rapid clearance of bacteremia. Convalescent, but not naïve, B1b cells generate a specific IgM response against B. hermsii, but the mechanism of IgM-mediated protection is unknown. Here, we show that neither Fcα/µR, a high-affinity receptor for IgM, nor IgM-dependent complement activation is required for controlling B. hermsii. Bacteria in diffusion chambers with a pore size impermeable to cells were killed when diffusion chambers were implanted into either convalescent or passively immunized mice. Furthermore, adoptively transferred convalescent B1b cells in Rag1(-/-) mice produced specific IgM that also cleared B. hermsii in diffusion chambers independent of complement. These results demonstrate that IgM-mediated clearance of B. hermsii does not require opsonophagocytosis and indicate that a mechanism for in vivo B1b cell-mediated protection is through the generation of bactericidal IgM.