Diversity of the Lyme disease spirochetes and its influence on immune response to infection and vaccination.

Synopsis: The Lyme disease spirochetes are a highly diverse group of bacteria with unique biological properties. Their ability to cycle between ticks and mammals requires that they adapt to variable and constantly changing environmental conditions. Outer surface protein C is an essential virulence determinant that has received considerable attention in vaccine and diagnostic assay development. Knowledge of OspC diversity, its antigenic determinants, and its production patterns throughout the enzootic cycle, as well as in the laboratory setting, is essential for understanding immune responses induced by infection or vaccination.

in N. America, whereas in Europe, all three species are present. Further exploration of the phylogenetic relationships among LD spirochete species and isolates led to the delineation of several additional species (55,57,65,67). The potential significance of these species in veterinary and human health remains to be defined.
The genus Borrelia has been recently divided into two genera; Borrelia and Borreliella (1).
Consistent with taxonomic precedent, since the TBRF species were described first they retain the Borrelia genus designation. The LD spirochetes and B. myiamotoi were assigned a new genus designation, Borreliella. Sharp differences of opinion exist concerning the practical implications of this reclassification (8,82). While the use of the designation Borreliella is voluntary, readers should be aware of this change as it has been fully applied in public databases and is beginning to appear in the literature.

UNIQUE FEATURES OF SPIROCHETES.
Spirochetes are distinct from other bacteria in several fundamental and fascinating ways. A feature shared by all spirochetes is their unique flat wave or spiral-like ultra-structure ( Figure 1A) (19). This characteristic morphology results from the presence of endoflagella, which are found in all spirochetes. The flagella arrangement in Treponema denticola, the periodontal disease pathogen, is shown in Figure 1B. The endoflagella are organized into two separate flagella bundles, each of which is anchored to the inner membrane at opposite ends of the cell. The flagella bundles sit within the periplasmic space and extend ~three quarters of the length of the cell (18).
Distinguishing features specifically of the LD and TBRF spirochetes include the composition of their cell wall and a unique genome arrangement (reviewed in (9)). Like Gram negative bacteria, they possess both an inner and outer membrane, but lack lipopolysaccharide (LPS). LPS is replaced by a diverse array of outer surface proteins (Osps) that play important roles in the host-pathogen interaction. Some of these Osps are described in detail below. The LD and TBRF spirochetes are distinct from all other bacteria, including other spirochetes, in that they possess a small, segmented genome consisting of a linear chromosome (0.9 Mb) and a series of linear and circular plasmids (7). Linear DNA is rare in bacteria. The plasmids range in size from 9 to 200 kb and comprise nearly 40% of the total genome (89). The total number and size of the plasmids carried by individual isolates can vary significantly (58). Plasmid variation results from plasmid loss, acquisition and genetic rearrangement (68). Some plasmids are dispensable (26), whereas others are essential for infection or survival (17). The unique properties of the LD spirochete genome are reviewed in (16).

DEVELOPMENTAL STAGES OF IXODES TICKS.
LD is maintained in nature in an enzootic cycle involving Ixodes ticks and a diverse array of mammalian reservoir hosts (42). The first developmental stage of a tick is the larva. Since transovarial transmission of the LD spirochetes in ticks does not occur, upon emerging from the egg, larvae do not carry the LD spirochetes. Ixodes ticks can only become infected by feeding on an infected mammal through a process referred to as Aacquisition.@ After taking their first and only bloodmeal, the six-legged larvae detach from their feeding source and molt into eight legged nymphs. This anatomical change has important implications for tick biology and feeding behavior, as it allows nymphs and adult ticks to climb up into brush where they gain better access to larger and more mobile mammals. Nymphs also feed just once and then molt into sexually differentiated adults. The body weight of adult female tick may increase by as much as 500 fold after the bloodmeal. Since male ticks do not feed, they play no significant role in transmission of LD. Images of engorged adult female I. scapularis and Amblyomma americanum (lone star tick) ticks are presented in Figure 2. It is important to note that pets and their owners are merely accidental hosts, and as such, do not play a significant role in maintaining LD in nature.

ADAPTIVE RESPONSES AND THEIR IMPORTANCE IN THE ENZOOTIC CYCLE
The acquisition of spirochetes by ticks and their transmission to mammals are active processes that are dependent on tightly regulated adaptive responses (43,75). Akins et al conducted a creative and pivotal study that provided insight into the nature of adaptive responses (3). They compared the protein content of laboratory grown spirochetes with that of host-adapted spirochetes. To accomplish this, cultures of LD spirochetes were placed in dialysis membrane chambers (DMC) and implanted in the peritoneal cavity of rats. Spirochetes maintained in DMCs become host-adapted and thus, more closely resemble spirochetes during natural infection (3). Comparison of the protein profiles of lab cultivated and host-adapted spirochetes revealed significant differences in the production levels of OspA, OspB and OspC (as well as other proteins). OspA and B were produced at high levels in lab cultivated spirochetes but not host-adapted spirochetes (3). In contrast, OspC production was low in laboratory spirochetes but high in host-adapted spirochetes ( Figure 3). The Akins study also proved central in shaping our understanding of humoral immune responses during infection. The Osp production patterns they reported are consistent with the development of a strong and early antibody response to OspC in mammals and the absence of a response to OspA and OspB (87). It is important to note that the low-level production of OspC during cultivation is well documented in the literature (62,88). Oliver et al demonstrated that only 10% of the individual cells in a laboratory culture produce detectable amounts of OspC (62).
Adaptation to the distinctly different environmental conditions present in unfed ticks, fed ticks, and mammals also requires changes in Osp production (23). In an unfed tick, spirochetes residing in the nutrient poor, midgut environment, produce high levels of OspA. Intake of a bloodmeal quickly changes the environment triggering a transition from OspA to OspC production (74). The upregulation of OspC at the tick-host interface is consistent with studies that have demonstrated that OspC is required for transmission and the establishment of an active infection in mammals. Strains that have been modified to not produce a functional OspC are unable to infect mammals (30,33,86).

OSP VARIATION: INFLUENCE ON VACCINE AND DIAGNOSTIC ASSAY DEVELOPMENT
The LD spirochetes produce a diverse array of Osps and the subset produced at any given time is controlled by environmental conditions. A comprehensive review of properties and functions of characterized Osps is beyond the scope of this report. While several Osps have been investigated for use in vaccine or diagnostic assay development (reviewed in (27)), discussion here is focused on OspC. OspC is a lipoprotein that varies in molecular weight (20 to 24 kDa) among isolates (38). The ospC gene is carried by a highly stable circular plasmid of 26 kDa (56) referred to as cp26 (86). An individual LD isolate produces only a single OspC protein variant. The antigenic diversity of OspC is well documented and has been intensively studied (32,45,85).
Prior to our current understanding of OspC phylogenetics, sequence variation seemed an insurmountable hurdle to overcome in efforts to employ OspC as a vaccine or diagnostic antigen (53). It was assumed that ospC variation arises through mutation during infection with subsequent immune selection allowing for the emergence of new antigenic variants. However, OspC is genetically stable during infection and thus and is not subjected to immune selection (81). Numerous distinct and stable variants of OspC, referred to as OspC Atypes,@ have been identified. OspC types are differentiated by a letter or other appropriate designation (reviewed in (53)). OspC proteins of a given OspC type are conserved with percent amino acid (aa) identity values of ~95% or greater. Identity values between OspC types can be as low as 65%. As an example, Earnhart et al compared the sequences of 55 OspC-type A proteins and found that amino acid identity values among these proteins were >97% (32) Studies by Brisson et al have provided significant insight into the biological rationale for the existence and maintenance of multiple, stable ospC types in nature (12,13). While an individual LD spirochete strain produces only a single OspC type, ticks commonly carry a heterogenous population of strains that as a whole can produce many different OspC proteins (25). The existence of multiple OspC types in a given tick may help to ensure that upon feeding, at least a subset of the strains can infect an animal that has been immunologically primed by previous exposure to other OspC types. It has been hypothesized that OspC type identity may also influence mammalian host compatibility (12). Certain OspC types may facilitate infection of specific mammals. Rhodes et al reported that the most common OspC type detected in infected canines was OspC type F (69). This is striking, as there have been no reports of the isolation of an OspC type F producing strain from humans. While more research is required to address the biological rationale for the maintenance of distinct OspC types in nature, OspC diversity is critical to consider when assessing host immune responses to this immunodominant early antigen.

IMMUNIZATION.
Evidence that antibody responses to OspC are type-specific came from studies in which mice were inoculated with individual LD strains producing different OspC types (28). Immunoblot analyses of the infection serum collected from these mice revealed that IgG responses are OspC type-specific. Rabbits immunized with purified recombinant OspC proteins also developed type-specific IgG responses (62). The lack of antibody crossreactivity with different OspC types is intriguing since segments of sequence are shared by all OspC proteins (i.e., are conserved). The specificity of the antibody response suggests that variable regions of OspC are presented to the immune system (62).
OspC-type specific antibody responses have also been demonstrated in naturally infected canines (62). Serum from dogs confirmed to be LD positive reacted with only a limited subset of OspC proteins in immunoblot analyses. The observed specificity of the OspC antibody response is consistent with epitope mapping studies that identified two dominant but variable epitopes of OspC (14). The regions corresponding to these antigenic domains were designated as the L5 and H5 epitopes. While the sequences of these epitopes vary among OspC proteins, they are highly conserved among proteins of an individual OspC type (14,29,32). The immunodominance of the L5 and H5 epitopes likely explains the basis for type-specific nature of the OspC antibody response.
It has been reported in some studies that a conserved motif of OspC drives antibody responses (46,52,70). This suggestion is difficult to reconcile in light of the type specific responses detailed above. This motif, referred to as either the C7, C10 or pepC10 motif, is proline rich and comprises the last 10 C-terminal residues of OspC (38). If a conserved sequence common to OspC types (i.e., C10) constitutes a dominant epitope, then antibody to OspC should bind to all OspC proteins. In this report, potential immune responses to C10 were further investigated. All methods employed in the experiments presented below have been previously detailed (62). Recombinant OspC proteins (type I, F and T) were generated with or without the C10 motif (OspC-IΔC10, OspC-FΔC10 and OspC-TΔC10), purified and screened with serum from representative LD positive horses (animal ID number: 1026) and dogs (TF1286). Serum from dog TF1286 bound to OspC type T but not to type F (Figure 4). Infection serum from horse #1026 bound to OspC type F but OspC type I. Furthermore, OspC proteins that lack the C10 motif were readily detected by antibody in infection serum. These observations support the contention that the C10 motif is not a dominant epitope and that it is the variable domains of OspC that drive antibody responses.

IMMUNITY?
It is common knowledge among veterinarians that practice in LD endemic areas that a significant percentage of dogs will develop repeated LD infections. This phenomenon is well documented in humans. In one study, 15% of LD patients living in a Lyme endemic area developed one or more follow up infections within 5 years (48). To add to our understanding of LD and protective immunity, we sought to determine if infection of mice with clonal populations of LD spirochetes results in broad, or strain specific, bactericidal antibody responses. In this report, separate groups of mice were infected with B. burgdorferiB31, N40 and 297 and B. afzelii PKo using previously detailed methods (30). Sera harvested from the mice were then tested for bactericidal activity against each strain using in vitro assays (44). Representative data are presented in Figure 5. Serum from mice infected with B. burgdorferiB31 efficiently killed B31 but did not kill B. burgdorferiN40, 297 or B. afzelii PKo ( Figure 5). Conversely, serum from mice infected with B. afzelii PKo efficiently killed PKo but not B. burgdorferi B31, N40, or 297. To determine if killing is complement dependent, one set of reactions were run with heat-inactivated complement or no complement added. Guinea pig serum (GPS) served as the exogenous complement source. No killing was observed unless active complement was included in the assay. The data indicate that serum mediated killing occurs through an antibody-mediated, complementdependent mechanism. More importantly, it can be concluded that infection with a given LD spirochete does not induce broadly protective antibody responses.

PREVENTION: THE KEY TO TACKLING THE LYME DISEASE PROBLEM.
Vaccination is widely considered to be the most cost-effective approach for prevention of infectious diseases. It stands to reason that concerns about accurate diagnosis and appropriate treatment strategies for LD could be alleviated to some degree through aggressive vaccination. Several licensed LD vaccines are available and approved for use in canines (27). These vaccines are of two general types: bacterin and subunit. Currently available bacterin vaccines are Novibac 7 Lyme (Merck), LymeVax 7 (Zoetis) and Ultra™ Durammune Lyme 7 (Elanco). Available subunit vaccines are VANGUARD 7 crLyme (Zoetis) and Recombitek 7 Lyme (Boehringer Ingleheim).

LYME DISEASE BACTERIN VACCINES
The composition of subunit and bacterin vaccines are inherently different. LD subunit vaccines consist of highly purified recombinant proteins (OspA and or OspC), whereas bacterin vaccines consist of lysates of two laboratory cultivated LD spirochete strains (20,49). The identity of the strains that comprise each commercially available bacterin vaccine is information that is not in the public domain. Because LD bacterin vaccines are generated from cell lysates, they contain a large number of proteins and other cellular constituents. In fact, genome sequencing and proteome analyses have demonstrated that the LD spirochetes can produce in excess of 1600 different proteins (10,37). The majority of these proteins are produced during laboratory cultivation (61). The precise proteins that are present in any given bacterin vaccine has not been reported. Importantly, most of the proteins produced by bacteria under any growth scenario are localized within the cell and function in metabolic pathways and other important cellular processes (71,72). While intracellular proteins can elicit an antibody response upon vaccination with a cell lysate based bacterin formulation, they are not likely to elicit Aproductive antibody@ (i.e., antibody that contributes to protective immunity), since in live cells intracellular proteins are not accessible to antibody. The removal of extraneous proteins from bacterins is conceptually beneficial as it would serve to direct and focus immune responses on immunologically relevant proteins.
The differential production of LD spirochete proteins under different environmental conditions (40) may also influence the composition and antigenic content of subunit vaccines. Since bacterins are made from cultivated bacteria, they may lack potentially protective antigens that are produced by the LD spirochetes only during residence in mammals (3). Similarly, there are additional proteins that are not produced during culture or in mammals that are selectively produced in ticks (15). Antigens that are produced during infection in mammals or ticks would intuitively be those that are most desirable for inclusion in a LD vaccine. In this context, subunit vaccines offer some advantages in that they are composed of carefully chosen antigens with known production patterns. In addition, subunit vaccines lack extraneous proteins that are not involved in triggering protective immunity (27).

LYME DISEASE SUBUNIT VACCINES
Recombitek7Lyme is a subunit vaccine consisting of lipidated OspA. Anti-OspA antibody inhibits transmission from ticks to mammals by targeting spirochetes in the tick midgut (36). OspA was also the sole component of LYMErix™ (SmithKlineBeecham), the only human vaccine to have made it to market (63). LYMErix™ was introduced in 1998 but then voluntarily removed in 2001. There were many factors that contributed to its demise and detailed assessments of its rise and fall can be found in several excellent reviews (60,90). Leaving the more controversial issues aside, LYMErix™ was compromised by low efficacy (49%) after a two-dose series. A three-dose series increased efficacy to 76% (80). The requirement for multiple boosts is due to the fact that OspA mediated protection is strictly dependent on high circulating antibody titers (80). If titers drop below a critical threshold level, spirochetes are able to transit into a vaccinated animal (24). Since OspA is not produced by the LD spirochetes in mammals (73), the spirochetes cannot be targeted by anti-OspA antibody after entering an OspA-vaccinated mammal. VANGUARD 7 crLyme (Zoetis), the newest canine LD vaccine to be approved by the USDA, is a subunit vaccine consisting of OspA and a recombinant chimeric OspC epitope-based protein referred to as a chimeritope. OspC chimeritopes consist of linear epitopes derived from several antigenically distinct OspC proteins that are joined together in a single recombinant protein (31). The rationale the behind the development of OspC chimeritopes was to generate a protein that can elicit antibody that can target OspC proteins produced by diverse strains. The conceptual rationale for chimeritope proteins has detailed in earlier reviews and hence is not discussed further in this report (27,53). Antibody elicited by OspC chimeritope vaccine antigens can target spirochetes during the process of transmission and during early infection in mammals. A vaccine that induces antibody that can kill spirochetes in both ticks and mammals has the potential to utilize two synergistic mechanisms of protection and thus be less dependent on the maintenance of high circulating antibody titers.

WHERE DO WE GO FROM HERE?
The Lyme spirochetes are a fascinating and remarkably diverse group of bacteria with unique biological properties. In this report, we have focused our discussion on the importance of understanding environmentally regulated protein production, the genetic and antigenic diversity of the LD spirochetes and how that diversity influences immune responses to infection and vaccination. There are many topics not addressed here that are equally worthy of discussion. As we move forward, our ability to critically assess and interpret the results of past and future studies focused on LD will directly impact how successful we are in addressing this important veterinary and human health concern.
Synopsis: The Lyme disease spirochetes are a highly diverse group of bacteria with unique biological properties. Their ability to cycle between ticks and mammals requires that they adapt to variable and constantly changing environmental conditions. Outer surface protein C is an essential virulence determinant that has received considerable attention in vaccine and diagnostic assay development. Knowledge of OspC diversity, its antigenic determinants, and its production patterns throughout the enzootic cycle, as well as in the laboratory setting, is essential for understanding immune responses induced by infection or vaccination. Izac   The unique structure of spirochetes. Panel A presents a dark-field microscopic image of Borrelia hermsii, a relapsing fever spirochete, in blood collected from an infected mouse.
Mice were infected by needle inoculation and blood samples were collected 3 days postinoculation. The characteristic spiral morphology shared by all spirochetes is readily visible. Note, there is high density spirochetemia. In contrast to relapsing fever, spirochetemias are not common in mammals infected with Lyme disease. Panel B presents a transmission electron micrograph of the oral spirochete Treponema denticola. The endoflagella bundles that are unique to spirochetes are indicated by the arrows. Izac    Specificity of the antibody response to OspC in infected dogs and horses. Recombinant OspC proteins (types I, F and T) with or without the putative C10 epitope were produced, purified and transferred onto membranes for immunoblot analysis. The membranes were screened with serum from an infected dog (ID #: TF1286; top panel) or an infected horse (ID #: 1026; bottom panel) and IgG binding to each protein assessed using the appropriate secondary antibody and chemiluminescence. Note that while antibody to OspC was detected, the antibody was not cross reactive with the different OspC types. In addition, deletion of the C10 motif from each protein had no discernable impact on the level of antibody binding. Izac Vet Clin North Am Small Anim Pract. Author manuscript; available in PMC 2019 July 01.

Figure 5.
Strain specific bactericidal antibody responses in mice infected with Lyme disease spirochetes. As detailed in the text, sera from mice infected with B. burgdorferiB31 or B. afzelii Pko were assessed for bactericidal activity. The infection sera were incubated with each strain with or without complement preserved-guinea pig serum (GPS). Bactericidal activity was measured by determining the percentage of spirochetes that were killed as a result of exposure to the infection serum (% killing). Note that the antibody mediated killing was strain specific and occurred through a complement dependent mechanism. It can be concluded that infection with an individual Lyme disease spirochete strain does not elicit a broadly protective antibody response. Izac Vet Clin North Am Small Anim Pract. Author manuscript; available in PMC 2019 July 01.