Cryptic and exposed invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi sl

Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies of B. burgdorferi sensu lato. IR(6) is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR(2) to IR(5)) were investigated. Antisera to synthetic peptides based on each of the IR(2) to IR(5) sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR(2), IR(4), and IR(5) but not to IR(3), indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.     

Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits

Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.     

An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi.

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable Ags such as the variable major protein of Borrelia hermsii, the variant surface glycoprotein of African trypanosomes, and the pilin of Neisseria gonorrhoeae include an immunodominant variable domain and one or more invariable domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain. VlsE (variable major protein-like sequence, expressed), the variable surface Ag of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR1-6) that together amount to half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Surprisingly, unlike the invariable regions of variable major protein, variant surface glycoprotein, and pilin, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane, as demonstrated by immunofluorescence and in vitro killing assays. VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. We submit that IR6 may act as a decoy epitope(s) and contribute to divert the Ab response from other, perhaps protective regions of VlsE.     

Serologic and mucosal immune response to rotavirus infection in the rabbit model.

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.     

Antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in white-footed mice

Serum samples were obtained from white-footed mice (Peromyscus leucopus) in tick-infested areas of Connecticut during the period 2001 through 2003 and analyzed for antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Emphasis was placed on the evaluations of highly specific recombinant VlsE or protein (p) 44 antigens of B. burgdorferi and A. phagocytophilum, respectively, in a newly developed enzyme-linked immunosorbent assay (ELISA) as well as testing sera with whole-cell antigens by conventional ELISA or indirect fluorescent antibody staining methods. Of the 414 mouse sera analyzed, 310 (75%) had antibodies to whole-cell B. burgdorferi, whereas 157 (38%) were positive to the VlsE antigen. The latter nearly equaled the overall antibody prevalence rate (37%) computed when sera were tested separately with the p44 antigen. Mice were exposed to these pathogens and B. microti (antibody prevalence = 25%) in extreme northern Connecticut as well as the southern coastal areas of the state, thus indicating further geographic expansion of these infections. Fifty-three (13%) sera from widely separated sites had antibodies to all three pathogens. With expression and immunological recognition of VlsE and p44 antigens in P. leucopus, separate incorporation of these fusion proteins in an ELISA was very helpful in confirming past or current infections and in identifying specific foci for B. burgdorferi and A. phagocytophilum.     

Spirochetes in ticks and antibodies to Borrelia burgdorferi in white-tailed deer from Connecticut, New York State, and North Carolina

Ticks were screened for spirochetes and serum samples from white-tailed deer (Odocoileus virginianus) were assayed for antibodies to Borrelia burgdorferi during 1983-1984. Using fluorescein isothiocyanate-labeled rabbit antibodies produced to B. burgdorferi, the etiologic agent of Lyme disease, spirochetes were detected in Ixodes dammini (10.5% of 1,193) and Dermacentor albipictus (0.6% of 157) adults from Connecticut, I. dammini nymphs (49.1% of 108) and adults (64.7% of 99) from Armonk, New York, and in I. scapularis (0.4% of 531) and Amblyomma americanum (3.5% of 173) adults from North Carolina. Infected ticks were either seeking hosts or feeding on deer during the summer and fall. Direct fluorescent antibody staining also revealed spirochetes in two larvae of I. scapularis that emerged from eggs deposited by separate females in the laboratory. Using indirect immunofluorescence tests, antibodies to B. burgdorferi were identified in white-tailed deer living in tick-infested areas of all three states. Aside from minor cross-reactivity, there was no serologic evidence of Treponema or Leptospira infections. Ixodes dammini is a primary vector of B. burgdorferi in northeastern United States, but in North Carolina, other ixodid ticks may transmit this spirochete to humans and wildlife.     

Changes in antigenic reactivity of Borrelia burgdorferi, the Lyme disease spirochete, during persistent infection in mice

Adult laboratory mice, Mus musculus, were shown to be suitable experimental animals for studying infectivity, persistent infection, and in vivo antigenic changes of Borrelia burgdorferi. Sixteen mice were inoculated intraperitoneally with a low-passage culture of an uncloned strain of B. burgdorferi and 16 months later spirochetes were reisolated from the urinary bladder of 15 (94%) of the mice. Spirochetes recovered from the urinary bladder of one persistently infected mouse were tested for infectivity and found to be infectious when passaged into four laboratory mice. Western blot analysis of immune serum from each of the persistently infected mice demonstrated that spirochetes used to infect the mice reacted differently when compared with the spirochetes subsequently reisolated from the mice, demonstrating for the first time that changes in antigenic reactivity had occurred in the spirochete populations during persistent infection.     

Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi.

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2(-/-) mice harbored up to 100-fold more spirochetes in tissues than did TLR2(+/+) littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2(-/-) mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2(-/-) mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2(-/-) mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.     

Genetic variation at the vlsE locus of Borrelia burgdorferi within ticks and mice over the course of a single transmission cycle

The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. The vlsE locus represents the best-studied example of antigenic variation in B. burgdorferi. During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population of B. burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts. Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles. Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.     

Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host.     

Susceptibility of the gray wolf (Canis lupus) to infection with the Lyme disease agent, Borrelia burgdorferi

Four juvenile gray wolves (Canis lupus) were inoculated with live Borrelia burgdorferi. One received an intravenous inoculum, a second was inoculated subcutaneously, and two more were fed Peromyscus maniculatus sucklings which had earlier been inoculated with B. burgdorferi. The intravenously inoculated wolf developed a generalized lymphadenopathy and a persistent serum antibody titer to the spirochete which peaked at 1:512. Borrelia burgdorferi was visualized in liver sections of this wolf using direct immunofluorescent staining. The subcutaneously inoculated wolf showed a low and transient antibody response which peaked at 1:64, and manifested no clinical or postmortem abnormalities. The wolves which were fed inoculated mice showed no detectable antibody response. They were clinically normal throughout the project, and there were no detectable lesions at necropsy. Two control wolves were inoculated intravenously with formalin killed B. burgdorferi. Serum antibody titers of these controls peaked at 1:64 and 1:32, respectively, and fell to 1:16 by day 48 postinoculation. A survey of serum samples from 78 wild-trapped wolves from Wisconsin and Minnesota revealed that one was positive and another was suspect for B. burgdorferi infection based on presence of antibody to the spirochete. We conclude that the wolf is susceptible to infection by B. burgdorferi and that wolves are being infected in the wild.     

Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.     

MyD88 plays a unique role in host defense but not arthritis development in Lyme disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88(-/-) mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88(-/-) mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88(-/-) mice responded poorly to Borrelia sonicate in vitro, MyD88(-/-) mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.     

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

Adaptation of Borrelia burgdorferi in the vector and vertebrate host

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, which afflicts both humans and some domestic animals. B. burgdorferi, a highly evolved extracellular pathogen, uses several strategies to survive in a complex enzootic cycle involving a diverse range of hosts. This review focuses on the unique adaptive features of B. burgdorferi, which are central to establishing a successful spirochetal infection within arthropod and vertebrate hosts. We also discuss the regulatory mechanisms linked with the development of molecular adaptation of spirochetes within different host environments.     

First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the Marbled polecat, Vormela peregusna and the European mink

ABSTRACT: BACKGROUND: Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease. CASE PRESENTATION: Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto. CONCLUSIONS: This is the first report of the Lyme disease spirochetes in the two mentioned hosts.     

Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation

Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.     

Variable VlsE Is Critical for Host Reinfection by the Lyme Disease Spirochete

Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.