Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale

Microbes and Infection, 2, 2000, 167−176 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

Review

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale Guy H. Palmer*, Wendy C. Brown, Fred R. Rurangirwa Program in Vector-Borne Diseases, Washington State University, Pullman, WA, 99164–7040, USA

ABSTRACT – Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence. © 2000 Éditions scientifiques et médicales Elsevier SAS ehrlichia / antigenic variation / persistent infection

1. Introduction Anaplasma marginale is the most prevalent tick-borne pathogen of animals worldwide, occurring in six continents and responsible for severe morbidity and mortality in temperate, subtropical, and tropical regions [1–3]. Initially described as a stage of the intraerythrocytic protozoan parasite Babesia bigemina, A. marginale was identified by Sir Arnold Theiler in 1908 as a distinct intraerythrocytic pathogen [4]. Paradoxically, this first identified ehrlichial pathogen was only recently recognized as a member of the tribe Ehrlichieae, following the application of 16S rRNA sequence analyses [5, 6]. This taxonomic reclassification has focused attention on the biological similarities between Anaplasma and Ehrlichia phagocytophila, the causative agent of human granulocytic ehrlichiosis [6]. This common biology is clearly evident in the importance of persistently infected mammalian reservoirs and ixodid ticks in pathogen transmission, which results in acute rickettsemia and clinical disease. In this review, we focus on the role of antigenic variation in maintaining persistent A. marginale infection and the transmission of these antigenic variants to initiate acute rickettsemia. Defining these events in A. marginale infection may provide the knowledge needed to improve control over this globally important livestock disease and, from a comparative medicine viewpoint, over the emerging ehrlichial pathogens of humans.

* Correspondence and reprints Microbes and Infection 2000, 167-176

2. Importance of persistent infection for tick-borne transmission A. marginale is transmitted by feeding of infected ixodid ticks on immunologically naïve cattle [1, 3]. The rickettsia invades mature erythrocytes and replicates intracellularly by binary fission. Infection becomes microscopically detectable when rickettsemia exceeds 107 organisms per mL of blood (figure 1). Acute signs of disease, characterized by severe anemia, occur as rickettsemia increases to > 109 mL-1 with 10–70% of all erythrocytes being infected [1, 3, 4]. Importantly, individuals that survive acute disease remain persistently infected with microscopically undetectable levels of < 107 mL-1 (figure 1). Rickettsemia levels during persistence are cyclical and vary from 102 mL-1 to 107 mL-1 (figure 1 inset) [7–9]. Persistent infection results regardless of the infecting A. marginale strain and has been demonstrated to persist without reinfection for seven years [7, 10]. This apparent lifelong persistence is fundamental to continued transmission as transovarial passage of A. marginale within the tick vector does not occur [11]. Consequently, maintaining a population of infected ticks for transmission is dependent on the infected animal reservoir. Acquisition of infectivity by ticks feeding on persistently rickettsemic cattle appears to be relatively efficient with over 50% of feeding Dermacentor andersoni adult male ticks becoming infected [10]. Although this tick infection rate correlates with the level of rickettsemia during acquisition feeding (figure 2), a substantial percentage of ticks, 27%, become infected even while feeding at the low levels of persistent rickettsemia that occur between the cyclic peaks [10]. Furthermore, the same number of A. marginale develop within the tick salivary 167

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Figure 1. Temporal course of acute and persistent A. marginale rickettsemia in cattle following tick transmission. gland at the time of subsequent transmission regardless of whether ticks acquisition fed on high or low points in persistent cyclic rickettsemia (figure 3). This efficient infection of feeding ticks and development of high levels in the salivary gland, coupled with the longevity of rickettsemia, support a critical role for persistent infection in continuous transmission. Thus, defining how A. marginale persists in cattle is directly relevant to understanding the key determinants of transmission.

3. Antigenic variation as a mechanism of persistent infection A. marginale persists in fully immunocompetent hosts [12]. Persistently infected cattle are protected

Figure 2. Relationship between mean A. marginale rickettsemia in persistently infected cattle and tick infection rate in adult male D. andersoni [10]. 168

against both high-level rickettsemia and clinical disease upon challenge with the homologous strain [1, 3, 12]. This paradox, in which the immune response effectively controls a challenge dose of > 108 ID100 but cannot clear a low-level persistent infection, suggests that persistence involves a mechanism of escape from the immune response. A. marginale infection of the mature erythrocyte eliminates the possibility that persistence is solely due to protection from the immune response within a long-lived host cell. The mean bovine erythrocyte life span of 160 days necessitates continual emergence and reinfection of new erythrocytes to maintain infection. These observations indicate that persistent infection must be a dynamic process characterized by continual invasion and replication.

Figure 3. Relationship between mean A. marginale rickettsemia in persistently infected cattle and organism number in infected adult male D. andersoni [10]. Microbes and Infection 2000, 167-176

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Figure 4. Presence of multiple msp-2 gene copies in the A. marginale genome [15]. A. marginale DNA from either the Florida (F) or South Idaho (I) strain was digested with restriction enzymes that cut outside of the msp-2 gene and Southern blotted. Restriction enzymes used are indicated at the top of each lane. Undigested bovine DNA was used as a negative control (BOV). Blots were hybridized with pCKR11.2 msp-2 (1,216bp; nt10-1226). The single copy gene msp-4 was used to control for multiple bands due to partial restriction enzyme digestion; there is a single HindIII site in msp4. Molecular size markers in kilobases are indicated on the left margin.

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Figure 5. Distribution of msp-2 copies in the A. marginale chromosome [15]. Clamped homogeneous electric field (CHEF) electrophoresis of SfiI digested A. marginale genomic DNA (F, Florida strain; I, South Idaho strain; V, Virginia strain) separates the chromosome into large, nonoverlapping fragments. Left panel: fragments were stained with ethidium bromide; the first two lanes contain lambda DNA/HindIII fragments and Delta 39 Promega Markers, respectively, as size markers. Right panel: fragments were Southern blotted and hybridized with pCKR11.2 msp-2. Molecular size markers in kilobases are indicated on the left margin. The identification of repeated cycles of rickettsemia, each composed of a progressive, logarithmic increase in organisms, followed by a precipitous decrease (figure 1, inset), led to the hypothesis that persistence reflected sequential emergence and immune control of antigenic variants [8]. Consistent with this hypothesis, the A. marginale variant types that compose the acute rickettsemia appear to be completely cleared concomitant with the onset of a primary immune response and distinctly different variant types emerge in persistent infection. Specifically, acute rickettsemia following tick transmission of the South Idaho strain of A. marginale is characterized by the expression of two distinct outer membrane proteins, the ARV1 and ARV2 variants of the major surface protein-2 (MSP2) [13]. However, the resulting persistent rickettsemia is composed of organisms, which express neither ARV1 or ARV2, but are characterized by newly expressed MSP2 variant types [13]. This complete clearance of the ARV1 and 2 variants and emergence of new sets of MSP2 variants within one week following control of acute rickettsemia supports the hypothesis that antigenic variation, rather than an ineffective immune response, is responsible for persistent infection. If sequential emergence of MSP2 antigenic variants is responsible for persistence, A. marginale must either contain numerous different msp2 genes or have a mechanism for continually generating additional diversity. This question is particularly interesting as the ehrlichial genomes are small, estimated at 1 250 kb for A. marginale [14]. All examined strains contain multiple polymorphic msp2 genes (figure 4), widely distributed throughout the chromosome (figure 5) [15]. Whether all these putative genes 170

express full-length proteins is unknown; however, transcription and surface expression of antigenically unique MSP2 has been confirmed for several gene copies [15, 16]. Sequence comparison of 25 full-length, expressed msp2 transcripts revealed the presence of a single hypervariable region characterized by nucleotide deletions, insertions, and substitutions resulting in structural polymorphism in the encoded MSP2 proteins [13, 17]. This central hypervariable region, represented by amino acids 180–275 (numbering based on the original genomic clone 11.2 MSP2), is flanked by highly conserved N- and C-terminal regions [9, 13, 17]. Importantly, the central hypervariable region is markedly hydrophilic, with 74% of the amino acids being hydrophilic, consistent with its surface exposure. In contrast, the highly conserved termini are composed of predominantly hydrophobic amino acids, consistent with membrane location. This single central hypervariable region, now identified in all of the 85 msp2 transcripts examined, appears to typify the variation in genogroup II ehrlichial pathogens as a similarly positioned, hydrophilic region has been identified in the E. phagocytophila msp2 homologue [17–20]. The relevance of the encoded MSP2 polymorphism to A. marginale antigenic variation and persistence is supported by the following observations: i) unique structural variants arise in each cycle of persistent rickettsemia; ii) variant-specific B-cell epitopes encoded by the hypervariable region are expressed in each cycle; and iii) specific immune responses to MSP2 are associated with organism clearance. As described above, South Idaho strain A. marginale expressing ARV1 and ARV2 MSP2 are cleared as acute rickettsemia is resolved, and the emergent organMicrobes and Infection 2000, 167-176

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Figure 6. Alignment of the predicted MSP2 amino acid sequences of the acute rickettsemia variant type ARV2 with the most similar persistent rickettsemia variant type, PRV1. Variant types were defined by reverse transcription (RT)-PCR of blood obtained from calf 767 following tick transmission of the South Idaho strain of A. marginale [13]. Areas of amino acid substitutions, insertions, and deletions are indicated by a white background, areas of amino acid identity have a black background, and grey shading indicates conservative amino acid changes. The dashes designate deletions. isms in early persistent infection express distinctly different MSP2 (designated PRV types 1–5). Sequence comparison of the hypervariable regions between the acute and early persistent rickettsemia variants revealed the expected variation, resulting in only 64–82% amino acid identity [13]. The sequence alignment of the most closely related acute and persistent rickettsemia variants, respectively, ARV2 and PRV1, is shown in figure 6. Furthermore, examination of a second time point, at four weeks of persistence, reveals emergence of a new set of MSP2 variants, distinct from those in both acute rickettsemia (58–72% identity) and the first time point of persistent rickettsemia (59–90% identity) [13]. Thus, the transition from acute rickettsemia to persistent rickettsemia and between time points in persistence are associated with clearance of MSP2 variant types and emergence of structurally distinct variants. This pattern continues, as shown by sequence analysis of later time points in these South Idaho strain infected cattle just described [13] and comparison of three persistent rickettsemia cycles in cattle infected with the Florida strain [17]. In each sequential cycle, newly expressed MSP2 variants emerge. The central hypervariable region encodes B-cell epitopes within the predicted surface domain [9, 16, 17]. Both monoclonal antibodies and polyclonal antibodies directed against the MSP2 hypervariable region have been shown to bind to variant-specific epitopes [9, 15–17]. Importantly, immunity against A. marginale involves antibody for both enhancing macrophage phagocytosis, and consistent with a role for MSP2 as an adhesin [21], direct neutralization [12]. Whether each MSP2 structural variant is antigenically distinct is unknown; however, variant-specific B-cell epitopes have been identified on two persistent rickettsemia variants despite higher than average identity (88%) within the hypervariable region [17]. Importantly, these Microbes and Infection 2000, 167-176

variant-specific epitopes are recognized by the infected animal during persistence and are bound by serum antibodies collected at the time when the rickettsemic cycle is controlled but not when the organisms expressing the variant first emerge at the start of the cycle [17]. While the temporal association between immune recognition of B-cell epitopes within the surface domain and the termination of a rickettsemic cycle suggests that variant-specific immunity is responsible for clearance of the replicating A. marginale, this critical point remains unproven. The present inability to grow and clone the intraerythrocytic stage of A. marginale has prevented standard in vitro approaches to test variant-specific neutralization. However, in vivo immunization and challenge studies support an important role for MSP2 specific immunity in control of A. marginale rickettsemia. Cattle immunized with purified outer membranes and completely protected from rickettsemia upon homologous challenge develop both high levels of MSP2 specific antibody and CD4+ MHC class II restricted T cells [22–24]. Notably, protection correlates with the anti-MSP2 titer and specific antibodies can block A. marginale binding to erythrocytes [21, 22]. More directly, immunization with purified MSP2 alone can induce complete protection against homologous strain challenge [25]. How well these immune responses, induced by immunization with purified protein and an adjuvant, represent the variant-specific responses that develop during acute and persistent infection is open to question. At present, the best evidence for MSP2 variant-specific immunity in A. marginale control during infection remains the complete clearance of specific variants composing the acute rickettsemia with only new variants emerging in persistence and the later repetition of this pattern of variant clearance and emergence during persistent rickettsemia [9, 13]. 171

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Figure 7. Alignment of predicted MSP2 hypervariable region amino acid sequences from salivary gland variant type SGV2 with the most similar acquisition feed variant type, AFV1. Acquisition feed variants were defined by RT-PCR of blood of calf 755, infected with the South Idaho strain, at the time of tick acquisition feeding. Salivary gland variants were defined by RT-PCR of salivary glands dissected from ticks at the time of transmission feeding to a susceptible calf. Areas of amino acid substitutions, insertions, and deletions are indicated by a white background, areas of amino acid identity have a black background, and grey shading indicates conservative amino acid changes. The dashes designate deletions.

4. Development within the tick restricts the MSP2 heterogeneity of transmitted A. marginale The population of MSP2 variants expressed in persistent infection is heterogeneous and, with time, continually changing. The specific variants that will be expressed do not appear to be predictable and individual animals, experimentally infected with the identical strain at the same time, express different variant types during each of the persistent rickettsemic cycles [9, 13, 17]. Furthermore, analysis of variants in consecutive rickettsemic cycles indicated that sequence heterogeneity increased over time during persistence [13]. As a result, ticks feeding during persistent infection ingest a heterogeneous population of variants that differ over time and among individual animals within a herd. Following this acquisition feeding, A. marginale undergoes a developmental cycle in the tick, starting with invasion and replication in the midgut epithelium and culminating in infectious stages within the salivary gland acini [26]. Strikingly, the heterogeneity of MSP2 variants in the acquisition bloodmeal is lost during development within the tick, and a restricted set of MSP2 variants are expressed in the salivary gland. The two salivary gland variants, designated SGV1 and SGV2 in the South Idaho strain, were distinctly different from the five variant types, AFV1–5, in the blood during tick acquisition feeding [13]. The amino acid identity in the hypervariable region between the most closely related variants, SGV2 and AFV1, was limited to 79% and is shown in figure 7. The other acquisition feed variants were even less closely related to SGV1 and SGV2, as shown in the phylogram of the encoded MSP2 amino acid sequences (figure 8). Only SGV1 and SGV2 are expressed in the salivary glands of D. andersoni ticks fed on cattle infected with the South Idaho strain—as shown by 100% nucleotide sequence identity of SGV1 and SGV2 in ticks that had acquired A. marginale by feeding on three different calves at different time points, feeding during both acute and persistent rickettsemia, and feeding on rickettsemic blood containing the distinctly different variant types [13]. The restriction of MSP2 variant heterogeneity in the salivary gland is significant as A. marginale expressing these variants are transmitted and subsequently compose the acute rickettsemia. Sequencing of 28 independently derived cDNA clones from acute rickettsemia following 172

transmission of the South Idaho strain reveals only two MSP2 variant types, designated ARV1 and ARV2, which are identical to, respectively, SGV1 and SGV2 [13]. Thus the restriction at the salivary gland level limits the antigenic heterogeneity of the challenge inoculum. This is in marked contrast to the tick-borne borrelial pathogens in which the outer membrane proteins switched on within the tick are no longer highly expressed following transmission to a new mammalian host [27, 28]. The transmitted A. marginale variants replicate in the immunologically

Figure 8. A phylogram of acquisition feed variant types (AFV) from calf 755, infected with the South Idaho strain, and subsequent salivary gland variant types (SGV) based on predicted amino acid sequences [13]. SEQBOOT, PROTDIST, PROTPARS CONSENSE programs in the PHYLIP-phylogenetic inference package [3.5] were used for the derivation of the data used in the phylogram [13]. Scale bar indicates 1% divergence in amino acid sequences. Bootstrap values from 100 analyses are shown at the branch points of the tree. Microbes and Infection 2000, 167-176

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naïve host, resulting in acute rickettsemia and clinical disease. Then, to complete the full circle of infection, the acute rickettsemia variants are cleared and persistent infection is established with the emergence of a new set of expressed MSP2 variants, each bearing a unique surface domain [13].

5. Addressing the unknowns of MSP2 antigenic variation Cyclic rickettsemia with sequential emergence and clearance of MSP2 antigenic variants are consistent features of A. marginale infection. However the proposed model of continuous generation of new MSP2 proteins bearing variant surface exposed B-cell epitopes may be an oversimplification [9, 29]. MSP2 occurs as both monomers and as multimers, predominantly tetramers, and unique variants can be simultaneously expressed. This may be relevant to those individual MSP2 variants that recur in more than one cycle. Although infrequent, composing only 13% of all variants [9, 13], these specific variants recur despite inducing a primary variant-specific immune response following their initial emergence [17]. Whether the quartenary structures derived from various combinations of different variants generate additional diversity in surface exposed epitopes and allow recurrence of an individual MSP2, now combined with new variants, is unknown. Alternatively, the recurrence may reflect variation in T-cell epitopes, outside the identical hypervariable regions. Single amino acid substitutions have been detected in the normally conserved regions flanking the surface exposed domain [16]. The detection of specific CD4+ T cells that recognize epitopes present on many but not all MSP2 proteins raises the possibility that T-cell epitope change occurs outside the hypervariable region ([24]; unpublished data). Both mapping T-cell epitopes and determining the effects of multimer structure on surface B-cell epitopes may be needed to better define how MSP2 variants evade the immune response. Similarly, the mechanism by which the multiple msp2 genes encode and express distinctly different MSP2 variants throughout persistence is unknown. A minimum of four MSP2 variants are expressed in each rickettsemic cycle and new cycles occur approximately every six weeks [9, 10, 15]. Thus over the seven-year period in which A. marginale has been shown to persist, greater than 240 different variants would be expressed. If each MSP2 is expressed from a single gene without recombination, this would require roughly 20% of the genome. Alternatively, recombination mechanisms could dramatically enhance the capability to express variants from a more limited number of polymorphic genes. Certainly, the presence of large, highly conserved regions flanking the hypervariable region provides the potential to generate diversity through gene conversion, and the presence of related blocks of insertions, deletions, and substitutions in different variants suggests that this mechanism occurs [9, 13, 17]. The complexity of the msp-2 gene family and potential for generation of new variants through recombination is dramatically enriched by the presence of the Microbes and Infection 2000, 167-176

Review

highly homologous msp-3 gene family. This multigene family, also identified in all examined strains of A. marginale, contains blocks of polymorphism, within and adjacent to the open reading frames, identical to sequences in msp-2 variants [30]. Consequently, the msp-3 gene family may serve as an additional mechanism to introduce polymorphism into MSP-2. Studies mapping expressed msp2 variant transcripts to the genome are currently under way and are expected to delineate the mechanism by which variation is generated and expressed. Sequence analysis of the genome and msp2 transcripts should also shed light on how expression is regulated. The restricted repertoire of variants expressed in the tick salivary gland could result from either selection from a larger population of variants acquired by the tick or, alternatively, de novo expression within the tick, as previously shown in Borrelia spp. [27]. Further evidence of regulation is the increased expression of salivary gland MSP2 when the tick attaches and initiates feeding [13]. This specific upregulation occurs concomitant with development of salivary gland stage infectivity and successful transmission [13, 31, 32]. Determining how specific gene copies are switched on and the signaling pathways that upregulate expression may provide new avenues for blocking required events for both persistence and transmission.

6. Relevance of A. marginale MSP2 antigenic variation as a model for other ehrlichial pathogens How appropriate is A. marginale as a model for other ehrlichial infections? While the mammalian reservoir of infection and the species of the ixodid tick vector vary among ehrlichial pathogens [6], the requirements for sufficient circulating organism numbers to infect ticks, replication and development of infectious organisms within the tick, and transmission feeding represent common features. Importantly, the small ehrlichial genome size limits the diversity of microbial mechanisms available to meet these common functions. The close genetic relatedness of organisms within this complex [5, 6] and the necessity of fulfilling these common transmission requirements provide a high likelihood that this group of pathogens uses similar mechanisms to enhance transmission. The initial infection of ixodid ticks occurs by feeding on a rickettsemic mammal, and duration of rickettsemia is a critical determinant of transmission. Like A. marginale, the other tick-borne ehrlichiae are transmitted either interstadially (between stages) or intrastadially (within a tick stage) but not transovarially [11, 26, 33, 34]. Consequently, maintenance of infected ticks in the field requires continual rickettsemic mammalian hosts. As noted previously, cattle infected with A. marginale remain rickettsemic for greater than seven years [7]. In addition, dogs carry E. canis at low levels for at least 34 months [35], and 100% of mice inoculated with E. phagocytophila became persistently infected [33], including white-footed mice, a proposed mammalian reservoir. White-tailed deer may also serve as a reservoir for E. phagocytophila and a 173

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significant percentage of deer in endemic regions are rickettsemic [36]. Similarly, E. chaffeensis rickettsemia persists for weeks after experimental infection, and there is a high incidence of rickettsemic deer in endemic regions [37–39]. Persistence therefore appears to be a common feature of tick-borne ehrlichial pathogens. Whether persistence is attributable to antigenic variation, especially in MSP2 homologues, in other ehrlichial pathogens is unknown. Homologues of A. marginale msp2 have been identified in additional members of genogroup II, A. ovis [40] and E. phagocytophila [18–20] and in the genogroup I pathogens, E. chaffeensis (designated omp-1) [41, 42], E. canis [41, 43, 44], and Cowdria ruminantium (designated map-1) [45, 46]. Each of these ehrlichial species uses a multigene family to encode structurally and antigenically distinct outer membrane protein variants that bear surface exposed, immunodominant B-cell epitopes. E. phagocytophila, the most closely related human pathogen to A. marginale, shares the common genetic feature of having the multiple msp2 genes widely distributed throughout the chromosome [47]. In addition, E. phagocytophila genes have a very similar central hypervariable region to encode polymorphism within the surface domain [17–20]. The E. phagocytophila and A. marginale MSP2 homologues are approximately 40–50% identical when compared as full-length proteins. However this comparison is skewed by the presence of the large central hypervariable regions. Excluding this central region, the identity between E. phagocytophila and A. marginale MSP2 homologues is 80–96% over the N-terminal third and 85–87% when the C-terminal thirds are compared [17]. Similarly to A. marginale, E. phagocytophila MSP2 is targeted by antibodies during natural infection [17–20] and encodes variant-specific B-cell epitopes, including neutralization-sensitive epitopes [48]. However, expression of different E. phagocytophila MSP2 variants [47], as well as variants of the group I ehrlichiae [43, 49, 50], has been shown only by comparison of strains and not, thus far, within a persistently infected reservoir host. Thus it is unclear whether the polymorphism in ehrlichial MSP2 homologues, other than those in Anaplasma, function solely as a mechanism of strain diversity or are expressed to allow persistence within the individual. Determining whether or not antigenic variants are sequentially expressed during persistent infection of the respective reservoir hosts is a key question to understand how broadly findings with A. marginale apply to other ehrlichial pathogens.

Acknowledgments This research was supported by NIH R01 AI44005, USDA 96–37204–3610, and USDA-BARD grant US-2238–92C. Figures 2 and 3 are based on data originally published in [10] and are represented with permission of the copyright holder, The American Society for Microbiology (ASM). Figures 4 and 5 were originally published in [15], and are republished with the permission of ASM. Figures 6 and 7 are based on data originally published in [13] and are represented with permission of the 174

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copyright holder, The Proceedings of the National Academy of Sciences (PNAS). Figure 8 was originally published in [13] and is republished with the permission of PNAS.

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