Approximately 20 closely related pathogenic and non-pathogenic species of Borrelia form the B. burgdorferi sensu lato complex. Of these 20 species, at least five are classified as causative agents of Lyme disease (US: B. burgdorferi sensu stricto; Europe and Asia: B. afzelii, B. garinii, B. spielmanii, B. bavariensis) (4–10). Lyme borreliae are carried and transmitted by several species of Ixodes ticks (Ixodidae, hard shell) though the most common species are I. scapularis and I. pacificus in the US and I. ricinus and I. persulcatus in Europe and Asia.

Species of Ornithodoros ticks (Argasidae, soft shell) carry and transmit relapsing fever spirochetes. Several Borrelia spp. cause relapsing fever but B. hermsii, B. turicatae, B. crocidurae, B. hispanica, B. duttonii are more commonly encountered.

While the general rule is Ixodes transmit spirochetes of the B. burgdorferi s.l. complex and Ornithodoros transmit relapsing fever borreliae, there are exceptions. B. recurrentis is a louse-borne relapsing fever spirochete endemic mainly to sub-Saharan Africa. B. theileri causes bovine borreliosis and is transmitted by Rhipicephalus microplus, a hard shell tick that parasitizes livestock (11). B. lonestari and B. turcica, spirochetes genetically similar to relapsing fever borreliae, are found in the hard shell ticks, Amblyomma americanum and Hyalomma aegyptium, respectively (12, 13). Finally, B. miyamotoi is a relapsing fever spirochete vectored by the same Ixodes spp. that transmit species of the B. burgdorferi s.l. complex.

In terms of disease, several tick-borne diseases are associated with non-specific symptoms (i.e., a possibly self-limiting “influenza-like” illness characterized by malaise, fatigue, aches, fever, and chills) (Table 1). While infection with Borrelia spp. generally results in similar symptoms, some species-specific symptoms can arise (14, 15). Erythema migrans and arthritis are commonly associated with a B. burgdorferi s.s. infection but rarely with B. afzelii infection, which more commonly manifests in the dermatological condition, acrodermatitis chronica atrophicans. B. garinii is more commonly associated with neurological symptoms. Relapsing fever is characterized by recurring spirochetemia corresponding to recurrent episodes of high fever not seen with B. burgdorferi s.l. infections.

Borrelia miyamotoi s.s. strains were first isolated and cultured in Japan in 1995 from I. persulcatus and the blood of Apodemus argenteus (small Japanese field mouse) (16). Since this initial isolation, B. miyamotoi DNA has been identified in I. scapularis, I. pacificus, I. ricinus, and I. persulcatus across the Northern hemisphere (17–84). B. miyamotoi DNA has also been identified in humans with a suspected tick-borne disease; while B. miyamotoi is associated with disease, teasing out the details of an infection with this spirochete has proven difficult for several reasons (85–92).

First, diagnoses based on serology can be problematic and lead to false-negative diagnoses. Several antigens, including 4 of the 10 assayed in a Lyme Western blot, are shared among Lyme, relapsing fever, and B. miyamotoi spirochetes (93, 94). Although Lyme and relapsing fever Borrelia cause different diseases and occupy different niches, species in this genus share a high degree of genetic homology (95–98). Therefore, some degree of cross-reactivity occurs between B. miyamotoi antibodies and B. burgdorferi s.l. antigens (91).

Second, an adequate and appropriate immunocompetent animal model to study B. miyamotoi infection is only now beginning to take shape. Without an optimal animal model to identify characteristic symptoms and pathologies, we are left to interpret and extrapolate symptoms from complex human cases where disease pathology can be complicated by underlying or unreported medical conditions or coinfections. Previous attempts to infect immunocompetent Peromyscus leucopus mice (a common reservoir for B. burgdorferi in the US) with B. miyamotoi s.l. LB-2001 (US strain) had been unsuccessful leaving severe combined immune deficient (SCID) mice as the only available animal model (17). SCID mice infected with B. miyamotoi exhibit sustained spirochetemia, similar to infection with relapsing fever spirochetes (99). Recently, however, Wagemakers et al. (100) were able to successfully infect immunocompetent C3H/HeN mice with LB-2001 and demonstrate spirochetemia 2 days post infection (dpi). Three of the eight mice infected exhibited relapsing spirochetemia at 5 and 6 dpi. More studies are required to determine the optimal animal model for B. miyamotoi infection (101–103).

Finally, B. miyamotoi’s status as a pathogen has only recently been established. The first confirmed human infections were reported in Russia in 2011 (85) with more cases subsequently described in the US, Europe, and Japan (86–91, 104–107).

Factor H is an ubiquitous 150-kDa soluble protein produced by diverse cell types throughout the human body (e.g., hepatic cells, fibroblasts, monocytes, endothelial cells) (132). FH consists of 20 short consensus repeats, while FHL-1 is a truncated variant of FH consisting of the FH N-terminal short consensus repeats 1 through 7. Both FH and FHL-1 are major direct regulators of the alternative complement pathway. In addition, FH and FHL-1 can directly regulate the classical and lectin pathways, though the regulatory roles in these pathways are minor compared to other classical and lectin regulatory mechanisms. Regulation is achieved through the recognition of self and non-self molecules via domains located on the C- and N-terminals, respectively (133–135). The C-terminal discriminates self from non-self through interactions with sialic acids, glycosaminoglycans, and sulfated polysaccharides, which are typically found only on host cells (136–140). FH binds self molecules with high affinity to prevent activation of complement. FH regulates the classical and lectin pathways by acting as a co-factor for FI. In this capacity, FH facilitates the serine protease activity of FI in cleaving and inactivating C3b. The alternative pathway is regulated through FH targeting factor Bb, which prevents the formation of fluid-phase C3 convertase and promotes decay (“decay acceleration activity”) of C3 and C5 convertases (141). For comprehensive reviews of FH and FHL-1, see Ref. (132, 141, 142).

Interactions with FH are the best-studied mechanism for Borrelia complement inactivation, and complement resistance is correlated with binding FH (143). Borrelia spp. bind FH and/or FHL-1 through various native proteins collectively termed FHBPs or CRASPs (125, 144, 145). CRASPs can be grouped by their ability to bind only FH or both FH and FHL-1 as well as the species specificity of binding (that is, whether a FHBP can bind FH from only one or several host species) (125, 145): CRASP-1 (CspA) and CRASP-2 (CspZ) bind both FH and FHL-1, while CRASP-3 (ErpP), CRASP-4 (ErpC), and CRASP-5 (ErpA) bind only FH. CRASPs bind soluble FH and maintain it in an active conformation thereby allowing FH to inhibit completion of the complement response (i.e., MAC formation).

Several relapsing fever spirochetes bind FH in vitro (125, 146–151). Two FHBPs, FhbA and BhCRASP-1, have been identified in B. hermsii strains YOR and HS1, respectively (152, 153). However, binding FH is not as important for relapsing fever spirochetes to establish infection as it is for Lyme disease Borrelia (154, 155). Further supporting the non-essential nature of binding FH, Woodman et al. (154) found that despite FhbA being surface exposed and strongly binding FH in vitro, only 16% of B. hermsii recovered from the blood of infected mice had detectable levels of bound FH.

C4b-binding protein (C4BP) has regulatory roles in all three pathways, though is the major regulator of the classical and lectin pathways. C4BP facilitates inactivation of C4b (classical, lectin) and fluid-phase C3b (alternative) by binding C4b, displacing C2a, and facilitating FI-mediated inactivation of C3 and C5 convertases (156).

Some Lyme and relapsing fever Borrelia spp. bind human and various animal C4BP (143, 148, 149, 157, 158). A comprehensive analysis identified outer surface proteins (Osps) associated with C4BP including OspA, Vlps, variable membrane proteins (Vmps), and several unidentified Osps (159). However, other studies have observed no binding of C4BP by Borrelia spp (143, 150, 160). These contradictory data may be due to differences in experimental design including the use of different strains, growth medium, temperatures, growth phases, and the use of recombinant versus native human C4BP. A putative C4BP receptor, p43, has been identified in B. burgdorferi s.l. (157). The relapsing fever spirochetes B. recurrentis and B. duttonii produce CihC, a surface lipoprotein homologous in sequence and function to fibronectin-binding proteins of other relapsing fever spirochetes, which also binds C4BP (148).

Resistance to complement is positively correlated to the infectivity of some Borrelia strains (130). With a higher resistance to complement, the more likely a bacterium can survive, disseminate, and proliferate. Co-opting tick proteins will protect spirochetes during the initial stages of transmission and dissemination but sustained dissemination requires Borrelia to resist complement via its own native mechanisms.

This leads to the question of how complement sensitive strains can cause infection. An interesting hypothesis was developed regarding complement resistance and spirochete niche when a relationship was noted between binding of the complement inhibitors, C4BP and FH (157, 158, 161). Neurotropic strains (e.g., B. bavariensis, B. garinii, B. turicatae, B. duttonii, and to a lesser extent B. hermsii) do not have to be highly resistant to complement in immunoprivileged sites, such as the central nervous system. Finding neurotropic species strongly bind C4BP and very weakly bind FH and FHL-1, while species that are not neurotropic bind C4BP but preferentially bind FH and/or FHL-1 supports this hypothesis (157). Alitalo et al. (162) did find B. garinii strains isolated from neuroborreliosis patients not only express FHBPs not expressed by strains cultured in vitro for an extended time but the FHBPs also bind FH. This implies complement resistance, though this was not reported and one of the isolates (LU59) was later reported to be highly but not completely sensitive to complement (163). It is possible that strong binding of FH is an artifact seen in vitro, similar to that observed with relapsing fever spirochetes (see FHBPs and CRASPs). Thus, binding FH is not required for neurotropic strains. Perhaps C4BP is sufficient to prevent complement activation during migration of neurotropic species from the site of inoculation to immunoprivileged sites. On the other hand, binding FH may be important for neurotropic strains to resist complement during migration and the incomplete sensitivity observed by Sandholm et al. (158) may be due to in vitro culturing resulting in the population losing its ability to bind FH. It could also be that neither C4BP nor FHBPs play a role in complement-sensitive borreliae disseminating and a novel mechanism is utilized by complement-sensitive strains.

Little information is available regarding the CD59-like protein of B. burgdorferi. Pausa et al. (164) demonstrated an increase in serum sensitivity and MAC formation in a serum-resistant B. burgdorferi isolate treated with anti-CD59 antibodies compared to the control treated B. burgdorferi and the serum-sensitive B. garinii isolate. In eukaryotic cells, CD59 is a surface-exposed membrane protein that prevents C9 polymerization and thus the formation of the MAC (19, 20). Still, it is not clear Borrelia possesses a protein homologous to mammalian or rodent CD59. While human anti-CD59 antibodies bound a surface-exposed integral membrane protein (29 kDa), this protein has never been identified though several known borrelial proteins can and have been ruled out based on molecular weight (e.g., BGA66, BGA71, OspA, OspB, OspC) (124). Given the demonstrated complement resistance conferred by this unknown borrelial protein, more attention should be given to identifying and clarifying the role this protein plays in complement resistance.

A large number of proteins with a vast array of functions have been identified in the saliva of feeding Ixodes spp. with more being identified and characterized (165–169). While the details and mechanisms for some of these proteins remain to be elucidated, the beneficial nature of Ixodes salivary proteins to spirochete transmission and survival has been established (170–177). Ixodes saliva contains adaptive and innate immunomodulatory and anticomplement proteins (165, 178–183). A recent study demonstrated changes in the salivary protein profile over the course of a feeding, which has implications for the efficacy of the host immune response at the feeding pit and for transmitting spirochetes (168). Currently, several members of the anticomplement family of proteins have been characterized from I. scapularis, I. ricinus, and I. persulcatus including Salp15, Salp20, Isac, Irac I, Irac II, and Ixac-B1, -2, -3, -4, -5.

Salp15 is able to inhibit both adaptive and innate immune responses (184, 185). Salp15 binds OspC, which both serum-resistant and serum-sensitive B. burgdorferi s.l. produce, to inhibit deposition of the MAC and block the recognition and binding of antibodies to OspC (172, 186–188). In addition, Salp15 expression increases when ticks are infected with B. burgdorferi (172). Interestingly, mice passively immunized with anti-Salp15 antibodies were protected from infection with B. burgdorferi (189).

Salp20 inhibits the alternative complement pathway through binding properdin, which prevents stabilization of C3 convertase and propagation of the alternative pathway (183, 190, 191). In addition, Salp20 enhances the activity of FH to inhibit the alternative pathway (183). Incubating a serum-sensitive B. garinii strain with Salp20 protected the strain from complement activation and lysis (190). The mechanism(s) by which Salp20 confer(s) protection to B. garinii is unknown.

The Isac-like family of proteins include Ixodes scapularis anticomplement (Isac), I. ricinus anticomplement (Irac I), Irac II, and Ixac-B1 through -5 (I. ricinus anticomplement). Proteins in this family are similar in function to Salp20 (180, 182, 192). Inhibition of the alternative complement pathway is achieved through targeting C3 convertase via interactions with properdin, as Salp20 does, and by preventing factor B from binding C3b or displacing factor B from C3 convertase.

Ornithodoros salivary gland extracts also possess proteins that inhibit the host immune response (193). To date, however, one complement inhibitor has been identified and characterized from one Ornithodoros spp. O. moubata, found in Africa, is the vector of the relapsing fever spirochete B. duttonii (194). O. moubata complement inhibitor (OmCI) is a lipocalin that binds to and prevents cleavage of C5 (195, 196). OmCI was found to be effective at inhibiting C5 cleavage in different mammalian and rodent hosts (196). It is unknown if OmCI protects B. duttonii or if homologous proteins are found in other Ornithodoros spp.

The Osps, particularly OspC, are one of the most studied group of Borrelia proteins. For comprehensive reviews of Borrelia Osps, see Ref. (145, 205, 206). OspE and OspF are discussed above with FHBPs. Less is known about OspA, a protein predominantly involved in uptake and survival in ticks. OspA is immunogenic and able to block antibody binding to another surface-exposed protein, P66 (207, 208).

OspC has diverse roles, many of which are essential for transmission from Ixodes and establishing infection in mammals (209–216). These studies were key in demonstrating that ospC is upregulated during the early stages of infection, downregulated after infection has been established, and deleting or overexpressing ospC results in spirochetes that are quickly cleared from a host.

A handful of immune evasion functions have been identified for OspC. As discussed above, OspC protects Borrelia by binding Salp15. OspC also prevents phagocytosis by macrophages (216). In addition, several OspC types have been identified and correlated with a strains ability to establish infection in hosts and reservoirs (217–222). However, as each Osp is present as a single-copy locus, genetic variation is seen at the population level. That is, outside of random mutation or horizontal gene transfer events, a single spirochete cannot produce different OspC types in situ.

In contrast, the Vls system can change the expressed surface antigen in situ (Figure 2). Antigenic recombination of VlsE is important in maintaining infection in mammals and helps Lyme Borrelia evade the humoral immune response (223–236). The Vls system is composed of approximately 16 vls cassettes (the exact number varies by strain) and one expression locus, vlsE. All of the identified vls cassettes are located on the same plasmid (lp28-1) in close proximity to but in the opposite direction of vlsE. Expression at vlsE occurs through the random recombination of segments of multiple vls cassettes rather than recombination of an entire, single vls cassette. Thus, recombination events result in thousands of unique VlsE variants, all approximately 36 kDa.

Variable membrane proteins, a system similar to Vls, are one of the best characterized immune evasion mechanisms (199, 237–240). B. hermsii has approximately 60 unique and promoterless vmp cassettes (i.e., silent cassettes) scattered throughout its genome and one promoter-driven vmp expression locus (Figure 2). A single vmp cassette is expressed when the entire cassette is moved to the expression locus.

The majority of spirochetes are cleared from the host through specific anti-Vmp IgM antibodies raised against the predominantly expressed Vmps, which results in a significant decrease in spirochete load (from approximately 10⁵–10⁷to <10⁴ spirochetes/mL). The remaining spirochetes consist of a small population expressing different cassettes. Since the host has not raised a strong antibody response to these non-dominantly expressed Vmps, this minority population of spirochetes can proliferate to high concentrations until an antibody response is mounted and the majority of spirochetes are once again cleared. This cycle of vmp conversion, peaking spirochete loads, and antibody-mediated clearing repeats a minimum of two times resulting in the characteristic symptoms of a relapsing fever illness.