As mentioned earlier, inhibition of the complement system is an important immune evasion strategy employed by many pathogens, including B. burgdorferi (57–59). B. burgdorferi proteins that interfere with complement activation allow for survival and dissemination of the pathogen from the initial site of infection (60).

The complement system consists of an evolutionarily highly conserved family of proteins that are found in all body fluids and serve three main functions during infection: trigger inflammation, opsonize pathogens, and form the “membrane attack complex” (formation of a pore in the cell membrane that causes cell lysis). Classical, alternative, and lectin are the three distinct pathways by which complement-mediated signaling and bacterial killing can be initiated [described in more detail in Ref. (61)]. Independent of the initial trigger, all pathways lead to the formation of a protease, the C3 convertase, which cleaves the complement component C3 into its activated components C3a (an inflammatory mediator) and C3b (an opsonin and immune stimulatory protein). The complement component C3b can also form the C5 convertase, another protease that cleaves complement component C5 into C5a and C5b, leading to the formation of the bactericidal “membrane attack complex” (61). C5a, like C3a, is a strong inducer of inflammation. During the process of complement activation, another complement component, C4, is cleaved into C4a and C4b. The latter also acts as an opsonin. Because of these multiple and highly pro-inflammatory effects, systemic activation of complement can cause septic shock if not appropriately regulated. Regulators of overshooting complement activation include components of the complement system itself: the Factor H family proteins and the C4b-binding protein. These proteins inhibit complement activation by a variety of mechanisms, including by accelerating the decay of C3 convertases, thus interrupting the complement activation cascade.

Borrelia burgdorferi has evolved complex mechanisms to evade complement-mediated killing by binding to the inhibitory host-regulatory factors [reviewed in Ref. (60, 62)]. B. burgdorferi expresses a diverse family of complement regulator-acquiring surface proteins, which recruit Factor H family proteins [reviewed in Ref. (63)]. Factor H and its relatives primarily inhibit activation of the alternative complement pathway. More recently, it was discovered that B. burgdorferi also binds to host C4-binding proteins, which primarily inhibit activation of the classical and lectin pathways (64). And, as outlined earlier, BBK32 seems to inhibit the classical pathway of complement activation via binding to the C1 complex (45). Thus, B. burgdorferi seems to target all three activation pathways of the complement cascade. The effects of complement inhibition on adaptive immune responses are outlined below.

The strong production of B. burgdorferi antigen-specific antibodies, as well as the strong increases in B. burgdorferi tissue load in SCID mice and B cell-deficient mice compared to wild-type controls (70, 71), has long been considered evidence of a robust and effective B cell response against B. burgdorferi infection. However, while the data provide clear evidence that B cells play an important role in the control of B. burgdorferi infection, this does not mean that these responses are optimally induced. Considering that B. burgdorferi infection results in persistent infections of mice despite these robust antibody responses, it is important to also consider the quality of the B. burgdorferi-specific antibody response. Several factors are known to affect the efficacy of humoral immune responses, including the epitope specificity of the induced antibodies, their immunoglobulin isotype profile, binding avidity to their cognate antigens, and various posttranslational modifications that can affect their effector functions (6, 20).

IgM is the first antibody isotype secreted during an immune response. It is important in controlling bacteremia and in activating the classical complement pathway. Immunoglobulin class switch recombination (CSR) to IgG typically occurs soon after an infection, and the four subtypes of murine IgG work together effectively to clear most pathogens (72). However, during B. burgdorferi infection, serum IgM levels remain high throughout infection (see text footnote). Moreover, hybridomas generated from lymph nodes of mice on days 8 and 18 postinfection showed that nearly half of B. burgdorferi antigen-specific cells were producing IgM, and the ratio of IgM to IgG never significantly changed throughout the infection (see text footnote) (73). The strong and ongoing production of IgM cannot be explained entirely by a predominance of T-independent responses, because depletion of CD4 T cells decreased the number of IgM-antibody-secreting cells (ASCs) (74). Thus, infection with B. burgdorferi induces an antibody response that is characterized by the continued production of IgM and IgG. Further studies will need to determine whether the strong production of IgM is evidence of a strong beneficial immune response or whether B. burgdorferi might be subverting the B cell response to generate this unusual Ig-isotype profile. Our studies have failed to find any beneficial effect of IgM on control of bacterial dissemination or B. burgdorferi tissue loads (see text footnote).

Given the important role of T cells in the regulation of CSR by B cells, the data may indicate a deficiency in T helper cell activation and/or functionality. In vitro data indeed provided some evidence that the CD4 T helper cell response induced by B. burgdorferi infection is distinct in function from that of CD4 T cells induced by immunizing mice with inactivated B. burgdorferi (75). T-dependent B cell responses usually also result in significant affinity maturation, i.e., an increase in the binding avidity of antibodies to their cognate antigens over time. Measuring antibody avidities of serum antibodies to a representative T-dependent antigen on B. burgdorferi N40, arthritis-related protein (Arp), however, failed to provide evidence for affinity maturation in the serum response to B. burgdorferi (75). Instead, we found that the binding avidity of the serum antibodies to Arp initially increased for the first 6 weeks of infection, but then peaked and steadily decreased thereafter to levels seen at the beginning of the infection. The rate of drop in antibody affinity was consistent with the normal half-life and decays kinetics of serum antibodies (75), suggesting that the ASC that generated the higher-affinity antibodies were short-lived.

Both CSR and hyperaffinity maturation of antibodies are T-dependent processes that usually occur in germinal center (GC) reactions in secondary lymphoid tissues. As we will outline below in more detail, we noted a collapse of the GC responses that coincided with the peak and then reversal of the antibody avidities. The data strongly suggest that the T-dependent GC responses are not fully functional during B. burgdorferi infection. The fact that immunization with inactivated B. burgdorferi infection resulted in robust GC responses suggests that exposure to live B. burgdorferi results in a subversion of the B cell response (73, 74). On the basis of these data, we propose that although present in large quantities, the B cell responses to and functionality of the serum antibody response to B. burgdorferi are suboptimal, enabling B. burgdorferi persistence, while also controlling B. burgdorferi tissue loads and thus overwhelming infection. Alterations in the B cell response quality and/or antibody functionality could provide a powerful immune evasion strategy for bacteria that are clearly susceptible to antibody-mediated immune clearance mechanisms (65, 66, 70, 71, 76, 77).

Although B. burgdorferi surface proteins have many functions in evading recognition by the host immune system, they are also antigens that trigger antibody responses. Additional immune evasion strategies therefore likely exist that inhibit recognition and antibody-mediated clearance of B. burgdorferi.

Borrelia burgdorferi is known to undergo major protein expression changes over the course of its life cycle, including expression of outer surface proteins. During transmission from the tick to a mammalian host, environmental signals trigger extensive global changes in gene expression (78, 79). Changes in surface protein expression also occur within a mammalian host during the course of infection. At least some proteins that trigger strong antibody responses are downregulated during the chronic phase of infection (80). One important example is OspC, a T-dependent antigen that is essential for initial colonization of mammalian hosts. Shortly after infection, this lipoprotein is no longer required, and if not downregulated triggers a strong and effective antibody response. However, expression is usually rapidly lost upon infection (81–84). This appears to be an effective immune evasion strategy, because constitutive expression of OspC prevents spirochete persistence, whereas bacteria that successfully downregulate OspC outcompete expressors in vivo (76, 85).

Antigenic variation is a process by which a pathogen varies the sequence of an expressed protein to avoid the deleterious effects of antibodies raised against it. Antibody-target switching has been demonstrated as an effective immune evasion strategy for numerous pathogens, including the closely related relapsing fever pathogen Borrelia hermsii (86–88). The B. burgdorferi genome contains the variable surface antigen E (vlsE) locus, which has received much attention as a major immunodominant surface protein of B. burgdorferi that undergoes extensive and rapid antigenic variation in mammalian hosts [reviewed in Ref. (89)]. In B. burgdorferi strain B31, VlsE recombination seems to be critical for B. burgdorferi persistence and the ability of B. burgdorferi to reinfect a host following antibiotic treatment (77, 90–92). It is interesting to note that vlsE recombination also occurs in antibody-deficient SCID mice, but not in vitro, suggesting that host triggers other than the antibody response direct this process (93). While it seems that a lack of variation of vlsE results in the rapid clearance of B. burgdorferi, it is less clear whether this process alone can explain the effective evasion of B. burgdorferi from antibody-mediated clearance. Recently, mathematical models have been put forward to suggest that a strongly immunodominant variable surface protein may prolong immune responses long enough to drive immune exhaustion (94), an intriguing idea that requires testing in the context of B. burgdorferi infection.

Finally, extensive genetic variation exists between the various Borrelia species and between individual bacteria (95–98). This allows adaptation to selective pressure from the host immune system, as well as to larger ecological niches, allowing strains to become fitter in a given geographic area (95). It also applies during the course of a chronic infection, as bacterial variants compete with each other (85, 99). Variation between strains is large enough in the context of the antibody response that different strains can infect the same mouse (100). Reinfection with the same strain has been tested in experimental settings after antibiotic treatment of mice. Studies by Piesman et al. and by Elsner et al. suggested that antibody-mediated protection wanes over time, even to the same strain of B. burgdorferi (69, 101). Given that long-term antibody production is usually induced following an infection, facilitated by the development of long-lived plasma cells that migrate to and then reside in the bone marrow (102, 103), the data further suggest that the antibody response to B. burgdorferi infection lacks some of these key components of successful humoral responses. It can be expected that each of the above outlined immune evasion mechanisms is unlikely to be solely responsible for persistence; instead they may act in concert. Whether these multiple apparent strategies represent redundant or synergistic effects remains to be established.

Modeling of population dynamics supports the establishment of equilibrium between B. burgdorferi and the host’s adaptive immune response (104). This is consistent with recent findings suggesting that the humoral immune response and, more specifically, the T-dependent B cell responses are a particular target of B. burgdorferi-induced immune suppression.

After escaping the site of infection, B. burgdorferi disseminates to other tissues via blood and lymph (43, 105). Mechanisms of entrance into the blood are better studied, but live B. burgdorferi is also capable of entering, persisting in, and traveling through the lymphatics (73, 105). Dissemination via the lymphatics has been described in other infections as an effective technique for immune evasion due to the specialized cell populations and altered immune milieu in these vessels compared to the bloodstream (106–108). Shortly after infection with cultured or host-adapted spirochetes (HAS), live B. burgdorferi enter lymph nodes draining the site of infection, resulting in massive lymph node enlargement (73, 109).

In secondary lymphoid tissues such as the lymph nodes, naïve T cells encounter antigens for the first time, and activated antigen-specific B cells receive the signals they need to proliferate. During B. burgdorferi infection, the presence of live spirochetes seems to interfere with these processes. Between days 5 and 7 postinfection of C57BL/6 mice with HAS, the separation of T cell zones and B cell follicles completely was lost within the draining lymph nodes. This degradation was not seen in mice that were inoculated with heat-inactivated spirochetes, suggesting that an active bacterial process causes the disruption of the normal immune response of the host (73, 74, 110). Interestingly, similar observations have been made also following infections with other pathogens, including infections with Plasmodium and Salmonella (111, 112). While during Salmonella infection lymph node architecture disruption was dependent on TLR4-mediated signaling (112), the observed changes during B. burgdorferi infection were independent of MyD88 and TRIF signaling (110). Given the importance of T and B cell trafficking within the lymph nodes for initiating and maintaining T–B interaction and immune response regulation, the data suggest that multiple pathogens, including B. burgdorferi, may have evolved strategies to interfere with the earliest processes of adaptive immune induction.

Furthermore, the presence of live (but not heat-killed) B. burgdorferi caused a disproportionate recruitment and proliferation of naïve follicular B cells, but not CD4 T cells, to the affected lymph nodes. This appeared to be a separately controlled process, but one also mediated specifically by live bacteria. B cell recruitment and/or retention required signaling through the type I interferon receptor by non-hematopoietic cells, likely lymph node stromal cell compartments (110). The resulting accumulation of large numbers of B cells in the lymph nodes explains the lymphadenopathy that is also observed in infected humans and dogs (113, 114). This pattern of lymph node architecture disruption followed by influx/retention of B cells is recapitulated later in more distant lymph nodes as they are infiltrated by the migrating spirochetes (73, 110).

The data support the concept that B. burgdorferi interferes with the induction and maintenance of adaptive immune responses by altering or hijacking innate immune signaling pathways. What effect does this have on persistence of B. burgdorferi? The organized lymph node structure and the regulated migration of lymphocytes within secondary lymphoid tissues is crucial for effective interaction of T and B cells and the induction of adaptive immune effector mechanisms. Disruption of this structure is liable to have long-term negative consequences for the quality of the immune response and thus increases the likelihood of pathogen persistence. We outline below evidence to this effect.

Activated B cells in the draining lymph nodes proliferate rapidly and initially form structures called extrafollicular foci (73). In extrafollicular foci, B cells become plasmablasts with or without T cell help and produce large quantities of B. burgdorferi-specific antibodies rapidly. B cells in extrafollicular foci, however, do not undergo extensive affinity maturation, nor do they form memory B cells or long-lived plasma cells (115). For this, GCs are needed. GCs are a complex and highly organized structures that form within the B cell follicles of secondary lymphoid tissues that consist of GC B cells, CD4 T follicular helper cells (T_FH), and follicular dendritic cells (FDCs). B cell activation and proliferation in GCs is facilitated by interaction with CD4 T cells and presentation of antigens on FDCs. High-frequency insertion of random mutations into the antigen-binding variable domain of the antibody molecule during this process, and the competition of the newly generated B cells for binding to antigen and for T cell-help, is thought to drive B cells with the highest affinity for antigen to outcompete other clones with weaker antigen binding. Although mechanisms are still unclear, GC responses lead to the development of two distinct cell populations: memory B cells, i.e., non-ASCs that circulate and can respond vigorously to repeat infection, and long-lived plasma cells, which continuously produce antibodies from their bone marrow niches and contribute to immune protection from reinfections (116–119). The GC environment also promotes CSR to IgG.

Despite the early presence of B. burgdorferi in the draining lymph nodes, GC B cell and T_FH numbers remain low for the first 2 weeks in experimentally infected mice (74) and then become measurable. However, although live B. burgdorferi remained present in the lymph node for at least 1 year after infection, and thus, B. burgdorferi antigens were presumably continuously available, the GCs collapsed around 1 month after infection, and associated B and T cell numbers decreased steadily over the next month (69). As outlined earlier, the decline in the avidity of serum antibodies against Arp follows the collapse of GCs (69).

Consistent with the short-lived nature of the GC responses, their functional ineffectiveness was demonstrated by experiments showing a complete lack of memory recall responses to both B. burgdorferi antigens and a co-administered vaccine antigen for many months after infection with B. burgdorferi. Stable continued antibody production by long-lived ASCs in the bone marrow was also strongly delayed for at least 3 months after infection (69, 74). The delay in these important B cell response outcomes is especially dramatic considering a mouse’s relatively short life span and likely frequent exposure in the wild. Although mice do not clear infection with B. burgdorferi, impairing the memory response could be advantageous to B. burgdorferi by leaving the host susceptible to secondary or superinfections. It might also prevent a timely and strong response to antigens that are dynamically upregulated and downregulated by B. burgdorferi as the infection progresses.

By decreasing the capacity of the host to produce effective antibodies against B. burgdorferi, the GC collapse may help B. burgdorferi evade clearance. The signals and mechanisms leading to the collapse, however, are unknown. One possible mechanism is the interference of B. burgdorferi with the complement system. Continued antigen presentation is crucial for hyperaffinity maturation, and components of the complement system are known to be involved in this process. Specifically, activated C3 and C4 fragments bound to antigen and adhere to complement receptors 1 and 2 (CR1 and CR2). These receptors are present on the major antigen-presenting cells in the GC, the FDC, and on GC B cells. It was shown previously that GCs will form normally in mice lacking CR1 and CR2, but collapse prematurely, before GCs can perform their important functions (120). This phenotype is strikingly similar to that seen in wild-type mice infected with B. burgdorferi. Interestingly, in B. burgdorferi-infected mice, although CR1 and CR2 are present on FDCs and GC B cells, C4 is not detectable (69). C4 is typically deposited on the surface of FDCs supporting antigen presentation. Interference with C4 deposition could inhibit antigen presentation by FDCs to GC B cells and thereby lead to GC collapse. B. burgdorferi interference with activation of complement could also have various indirect effects on GCs: changing the cytokine milieu, reducing antigen presentation to naïve B cells via CR1 on APCs outside the GC, reducing naïve B cell activation via co-stimulation with CR2, and reducing opsonization (and thus uptake) of antigens. Exploring the role of complement and complement inhibition by B. burgdorferi during infection are important subjects for future studies.