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Infection and Immunity, November 2007, p. 5185-5190, Vol. 75, No. 11
0019-9567/07/$08.00+0     doi:10.1128/IAI.00913-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Maintenance of Antibody to Pathogen Epitopes Generated by Segmental Gene Conversion Is Highly Dynamic during Long-Term Persistent Infection ^,

Yan Zhuang, James E. Futse, Wendy C. Brown, Kelly A. Brayton, and Guy H. Palmer^*

Programs in Vector-Borne Diseases and Immunology, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington

Received 5 July 2007/ Returned for modification 14 August 2007/ Accepted 17 August 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Multiple bacterial and protozoal pathogens utilize gene conversion to generate rapid intrahost antigenic variation. Both large- and small-genome pathogens expand the size of the variant pool via a combinatorial process in which oligonucleotide segments from distinct donor loci are recombined in various combinations into expression sites. Although the potential combinatorial diversity generated by this segmental gene conversion mechanism is quite large, the functional variant pool depends on whether immune responses against the recombined segments are generated and maintained, regardless of their specific combinatorial context. This question was addressed by tracking the Anaplasma marginale variant population and corresponding segment-specific immunoglobulin G (IgG) antibody responses during long-term infection. Antibody was induced early in A. marginale infection, predominately against the surface-exposed hypervariable region (HVR) rather than against the invariant conserved flanking domains, and these HVR oligopeptides were most immunogenic at the time of acute bacteremia, when the variant population is derived via recombination from a single donor locus. However antibody to HVR oligopeptides was not consistently maintained during persistent infection, despite reexpression of the same segment, although in a different combinatorial context. This dynamic antibody recognition over time was not attributable to the major histocompatibility complex haplotype of individual animals or use of specific msp2 donor alleles. In contrast, the position and context of an individual oligopeptide segment within the HVR were significant determinants of antibody recognition. The results unify the genetic potential of segmental gene conversion with escape from antibody recognition and identify immunological effects of variant mosaic structure.

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Bacterial and protozoal pathogens that establish persistent infection by sequential generation of antigenic variants most commonly rely on gene conversion events that recombine complete or partial donor sequences into active expression sites (3, 7, 8, 10-12, 22, 29, 30). The taxonomic diversity of pathogens that utilize gene conversion, from alphaproteobacteria and spirochetes to apicomplexan protozoa, and the over-20-fold range in their genomic capacities illustrate the broad utility of this basic mechanism (23). Both large- and small-genome pathogens use a combinatorial mechanism in which unique donor oligonucleotide segments can be recombined in different orders and combinations to generate a tremendous number of potential variants—from hundreds for bacteria to millions for the large-genome African trypanosomes (8, 23). Despite the broad utility of segmental gene conversion, the immunologic consequences of this mechanism remain largely unexplored, and thus there is a major gap in knowledge as to whether the potential combinatorial sequence variant pool realistically represents an actual antigenically variant pool. Specifically, if the immune response recognizes and maintains memory for epitopes encoded by an individual recombined oligonucleotide segment regardless of its combinatorial context, this would dramatically reduce the number of true antigenic variants compared to the number of potential variants generated by segmental gene conversion.

To date, this question has been difficult to address due to both a lack of complete knowledge of the potential variant donor sequence repertoire and an insufficient collection of sequential antigenic variants to track segmental usage along with the immune response over time. We approach this question by study of Anaplasma marginale during long-term persistent infection in calves, a natural ruminant reservoir host. A. marginale is a prototypical antigenically variant bacterial pathogen that establishes persistent infection in the bloodstream and evades clearance by sequential emergence of distinct surface variants (24). The variation primarily occurs in the immunodominant major surface protein 2 (MSP2) within an extracellular domain, the hypervariable region (HVR) (13-15). Unique variants are generated by gene conversion events in which the complete expression site HVR or an oligonucleotide segment within the expression site HVR is replaced using chromosomal donor sequences, termed MSP2 functional pseudogenes (Fig. 1) (7, 8). The St. Maries strain of A. marginale contains five distinct chromosomal msp2 functional pseudogenes and a single expression site (6). The pseudogene sequences represent essentially the full repository for variant generation, as the only other mechanism, mutation associated with mismatch repair, accounts for only approximately 2% of the variation (16). Consequently, this afforded the opportunity to examine the development and maintenance of the antibody response against the full potential set of recombined oligopeptide segments during long-term A. marginale persistent infection. In the present study, we utilized a large data set, in which the specific recombined segments of >600 MSP2 variants were identified during persistent infection and immunoglobulin G (IgG) antibody binding to the encoded polypeptides was tracked using >700 individual binding assays to resolve whether the potential combinatorial sequence variant pool represents an equivalent antigenically variant population.

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FIG. 1. A. marginale MSP2 expression site and HVR structure. (A) Complete repertoire of potential HVR segments encoded by the five unique msp2 donor pseudogenes of the Anaplasma marginale St. Maries strain. (B) Oligopeptides representing the conserved N- and C-terminal expression site domains. These are conserved in all MSP2 variants regardless of the recombined HVR. (C) Unique msp2 expression site variants are generated by gene conversion. An intact donor pseudogene can recombine into the expression site or, as occurs during persistent infection, sequential rounds of recombination generate complex expression site mosaics encoding HVR oligopeptides from different pseudogene donors. Specific sites of recombination are indicated with an "X." The chromosomal positions of the pseudogenes and single expression site are not shown to scale, and the duplicated pseudogenes are not shown. The complete genome sequence with annotated pseudogenes and expression site has been reported previously (6).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Variant tracking during A. marginale infection. The 613 MSP2 variants in the study were derived from a larger set of 1,333 variant sequences previously reported (16). Briefly, infection was initiated in each of four individual Holstein calves (983, 985, 990, and 995) by transmission feeding of Dermacentor andersoni infected with the St. Maries strain of A. marginale. The variant populations at peak bacteremia (10⁹ organisms per ml) and at 4, 6, 8, and 11 months postinfection (persistent bacteremia; bacteremia at all time points was 10⁷ organisms per ml) were determined by specific amplification of the single msp2 expression site and sequencing the expression site copy as previously described in detail (7, 16). A minimum of 30 clones were sequenced at each time point, an approach that provides a 95% confidence level that any variant present at a frequency of >10% in the population will be detected and that allowed determination of the proportional representation of each specific variant within the oligoclonal variant population (28). Each expression site HVR sequence was mapped either in its entirety to one of the five unique msp2 functional pseudogenes or as individual segments to its respective donor pseudogene (16) using the complete genome sequence of the St. Maries strain (GenBank accession no. CP000030). The composition of the expression site HVR for each of the 613 variants was denoted by the oligopeptide corresponding to each segment in an N-terminal-to-C-terminal direction (Table 1).

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TABLE 1. Loci, designations, and sequences of the oligopeptides representing the complete repertoire of potential HVR segments

Measurement of HVR segment-specific antibodies. The complete repertoire of HVR segments encoded by five unique msp2 functional pseudogenes was represented by 18 overlapping 30-mer synthetic peptides (Fig. 1A; Table 1). The invariant N- and C-terminal MSP2 domains were represented by an additional 16 overlapping 28- to 30-mer peptides (Fig. 1B; Table 2). Sera were obtained from each infected animal within 2 weeks following each time point when the variant composition was determined by sequencing the expression site HVR. Immulon II 96-well enzyme-linked immunosorbent assay (ELISA) plates were coated with 1 µg of peptide per well in coating buffer (50 mM Na₂CO₃, pH 9.6) overnight at 4°C, washed with phosphate-buffered saline (PBS) containing 0.05% (vol/vol) Tween 20, and then blocked with PBS containing 5% (wt/vol) milk and 0.05% (vol/vol) Tween 20 for 1 h. Sera were diluted in blocking buffer, and 50 µl/well was added to triplicate wells. Following washing, 50 µl of a 1:500 dilution of recombinant protein G-horseradish peroxidase (Zymed) was added per well, and the plates were incubated for 1 h at room temperature. After additional washes, binding of protein G was detected using KPL SureBlue microwell peroxidase substrate at 100 µl/well for 15 min and then stopped with 100 µl of 1% hydrochloric acid. The optical density at 450 nm (OD₄₅₀) was determined for triplicate samples, and positive binding was defined statistically as exceeding the mean plus 3 standard deviations of the OD₄₅₀ of preinfection serum from the same animal and exceeding three times the absolute mean value of the OD₄₅₀ of the test serum with the control peptide P1 (which was more than the mean value at OD₄₅₀ plus 3 standard deviations). All sera were tested at a minimal dilution of 1:10 before being classified as negative for a specific oligopeptide segment. Antibody to the invariant protein MSP5 was measured by ELISA as previously described in detail (32).

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TABLE 2. Loci, designations, and sequences of the oligopeptides representing the conserved expression site domains

Determination of major histocompatibility complex class II (MHC-II) haplotypes. The bovine lymphocyte class II haplotypes were determined by PCR-restriction fragment length polymorphism analysis of exon 2 as previously described (9) and classified as defined previously (19). The class II DRB3 haplotypes were as follows: animal 983, 22/23; animal 985, 16/22, animal 990, 23/24; animal 995, DRB3 8/22.

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The IgG antibody response is preferentially directed against the MSP2 HVR and is highly dynamic during persistent A. marginale infection. The MSP2 variant population was identified during acute bacteremia and throughout persistent infection in each of four animals. Initially we confirmed that each potential HVR segment (Fig. 1A) was indeed expressed in each animal by examination of the sequenced expression site variants at each time point. Each HVR segment was expressed at a minimum of three different time points, including at least two during long-term persistent infection. We then determined when IgG to each HVR segment and the conserved domains first arose and whether these antibodies were maintained during persistent infection. The highest percentage of HVR-specific antibodies was detected at the time of acute infection, during which antibody to 81% ± 13% of the individual HVR oligopeptides was detected. This is consistent with prior observations that a strong IgG antibody response develops at the time of peak A. marginale bacteremia and is associated with initial control of acute infection (21, 26). If this HVR segment-specific antibody response were to be maintained and expanded, this would be expected to limit the utility of segmental gene conversion as an escape mechanism. However, the percentages of segment-specific antibodies to the HVR oligopeptides during persistent infection were significantly lower (P < 0.05): 43% ± 16%, 39% ± 10%, and 39% ± 15% at 8, 11, and 24 months of infection, respectively (Fig. 2). Antibody to only 28% of the individual HVR oligopeptides was continuously maintained throughout persistent infection compared with 62% for the HVR oligopeptides which were recognized by antibody at some time points but not consistently. This dynamic antibody response was specific to the HVR segments, as indicated by the induction of antibody to an invariant diagnostic antigen (MSP5) (18, 32) at the time of acute bacteremia and maintenance of antibody binding at all time points in all animals. In contrast, IgG antibody reactivity to the conserved domain MSP2 peptides was very limited: no peptide induced antibody in all persistently infected animals despite continual exposure, and only one peptide (P14) was recognized by antibody at all time points in a single animal (C983).

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FIG. 2. Maintenance of segment-specific antibody during long-term persistent infection. The percentages (means ± standard deviations) of the total HVR segmental repertoire that were recognized by antibody obtained throughout infection are shown. Antibody to the invariant MSP5 was present at all time points in all animals (data not shown). Results represent the means for the four animals (C995, C990, C983, and C985) at each time point, except for the 11th and 24th months, each of which represents the mean for three animals (no serum was available for animal C985).

The dynamics of segment-specific IgG antibody do not correlate with expression of segments within the HVR. To test whether maintenance of segment-specific antibody during persistent infection directly reflected the frequency and level of HVR segment expression, we calculated the proportional representation of each segment within the oligoclonal population of variants at each time point and compared this to the magnitude of the segment-specific antibody response (see Table S1 in the supplemental material) using partial-correlation analysis (34). Partial-correlation analysis allows assessment of the strength of the relationship between two variables (in this case, segment expression and antibody binding) after removing the effects of other variables (in this case, the individual animal, peptide, and time) that may generate spurious correlation or mask true correlation. Although there was strong correlation for specific segments in the variant population, there was no overall agreement between proportional usage of the HVR segments and antibody reactivity (Table 3). This lack of agreement held true when the segmental repertoire of each msp2 donor pseudogene was examined separately or when the complete segmental repertoire was analyzed as a group (Table 3). This relationship was observed during infection of each individual animal and was independent of MHC haplotype (Table 2). These results indicated that the dynamic response was not simply linked to recent antigen exposure but suggested that the progression of segmental combinatorial diversity during infection, which generates increasingly complex MSP2 mosaics (16), was involved.

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TABLE 3. Partial correlationsa for the relationship between proportional representation of segments in expressed variants and the presence of segment-specific IgG antibody

The dynamics of segment-specific IgG antibody are linked to position within and composition of the expressed HVR. We hypothesized that the dynamic antibody response to specific oligopeptide segments was attributable to the context of the segment within the expressed HVR. This hypothesis is consistent with recognition early in infection, when segments are presented in the context of an intact recombined pseudogene donor (7, 16, 28), but with variable recognition during long-term persistent infection, when segments are recombined in a mosaic fashion with segments derived from distinct pseudogene donors (16). First, we identified the positions within the HVRs of those oligopeptide segments to which antibody was developed and maintained (28% of the total segmental repertoire) versus those to which antibody was dynamic during infection (62% of the total repertoire). Each segment was assigned a position within the HVR as N terminal, N middle, C middle, or C terminal. Maintenance of antibody throughout infection was associated with position within the HVR, as 50% of the segments in the N terminus were uniformly bound by antibody versus only 6% at the HVR C terminus (Table 4). This pattern was consistent regardless of the specific donor pseudogene. We then asked whether the HVR context of an individual segment, either flanked by segments derived from the same donor pseudogene or within a mosaic, was associated with the presence or absence of segment-specific antibody. Two contiguous segments from donor pseudogene 1, I.1 and I.2, were selected for analysis based on the following criteria: (i) they were recognized by antibody obtained at the time of acute infection and thus were identified as being immunogenic; (ii) antibody binding during persistent infection was not consistently linked to expression during persistent infection; (iii) there were a sufficient number of variants in which each segment was expressed, either in the context of its donor pseudogene or as a mosaic with specific C-terminal segments, to provide statistical power; and (iv) the analyzed variant population was distributed among animals with distinct MHC types. Importantly, the absence of antibody binding to these two segments, I.1 and I.2, despite their high proportional representation in the total variant population at specific time points in each animal (Fig. 3), demonstrated that the lack of association between segment expression and antibody response cannot be attributable to inaccurate sampling of the variant population. Throughout persistent infection, the lack of antibody against these oligopeptide segments was significantly associated with the presence of specific C-terminal segments in a mosaic structure as opposed to the development of antibody when segments I.1 and I.2 were present within the context of their donor pseudogene (Fig. 4). This association was statistically significant (P < 0.001) for both segments I.1 and I.2.

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TABLE 4. Relationship of the position of an individual segment within the HVR and maintenance of antibody reactivitya

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FIG. 3. Lack of association between expression of 1 segments I.1 and I.2 and antibody (Ab) response during persistent infection. Segment-specific antibody binding (represented by the OD450 value) is represented on the left axis, and proportional representation in the total variant population is represented on the right axis. Results are shown for each individual animal (C995, C990, C983, and C985) from preinfection (P) to the 11th month after the tick transmission (months 1 and 11 are missing for animal C985).

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FIG. 4. Effect of context within the HVR on the immunogenicity of 1 segments I.1 and I.2. Bars represent the proportions of variants (means ± standard deviations) containing segments I.1 or I.2 in the presence of I.3, I.4, V.3, or V.4, as mosaics near the C terminus of the HVR, when specific antibody (Ab) to I.1 and I.2 is present (black bars) or absent (hatched bars). Three continuous segments in the order of their original donor pseudogene sequence, such as I.2-I.3-I.4 or I.1-I.2-I.3, are not considered as mosaics.

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This large data set of 613 variants and 748 antibody binding analyses throughout persistent infection provides a link between the molecular mechanism of generating novel variants via segmental gene conversion and immune evasion. The overlapping HVR oligopeptides used in this mapping study were selected to encompass the complete donor repertoire (6) and to represent the mean oligopeptide length (29 ± 13 amino acids) encoded by msp2 segments recombined during persistent A. marginale infection (16). While these oligopeptides cannot represent all conformational B-cell epitopes created by unique HVR combinations, MSP2 oligopeptide epitopes have been previously shown to be surface exposed, and development of variant-specific peptide IgG antibody has been shown to be associated with variant clearance (13-15). Thus, induction and continual maintenance of antibody to these epitopes would preclude later reuse of the segment, which is required to generate the number of MSP2 variants theoretically required for long-term persistent infection and actually identified in vivo (8, 16). While A. marginale is limited by its small genome size (1.2 Mb) in its capacity to encode full-length variants and thus is highly dependent on the combinatorial diversity generated by segmental gene conversion, a very similar mechanism has been shown to occur in African trypanosomes despite the much greater genomic capacity (4, 5, 30). Trypanosoma brucei and Trypanosoma equiperdum have both been shown to express variable surface glycoproteins (VSG) derived from a mosaic of different vsg segments recombined into an expression site (17, 27, 31). Whether the same VSG oligopeptide is commonly expressed in different contexts, encoded as part of a full-length recombined vsg and later as a segment within a vsg mosaic, is currently unknown and awaits completion of the genome sequence and tracking of a larger repertoire of variants over time. However, one may hypothesize that, as is found for A. marginale, frequent reappearance of an immunogenic VSG segment would be in the absence of continuously maintained specific antibody.

Individual MSP2 HVR segments were most immunogenic early in A. marginale infection, when they are expressed in the context of the complete pseudogene. This may represent a structural effect on immunogenicity or, not mutually exclusive, an effect of the higher bacteremia levels during acute infection. We have recently shown that MSP2 variants with HVRs derived from a single donor pseudogene, designated simple variants, have a marked in vivo fitness advantage compared to complex variants with mosaic HVRs (25). Combined with the present data, this suggests that there is an association between the MSP2 HVR structure and both in vivo growth fitness and immunogenicity. Thus, simple variants have a clear but, due to their immunogenicity, short-lived in vivo fitness advantage. In contrast, the complex variants are at a fitness disadvantage (2 to 6 log₁₀ fewer bacteria) compared to simple variants during acute bacteremia (24) but may compensate with lower immunogenicity and long-term persistence. Specifically, there was significantly less antibody recognition of N-terminal epitopes when the epitope-bearing segment was recombined in a mosaic with flanking regions derived from different msp2 donor pseudogenes. As N-terminal segments are the most immunogenic early in infection, this suggests that the context can produce a marked effect on intrinsic immunogenicity and is consistent with repeated reappearance of a segment in a new HVR context.

The immunologic mechanism underlying this dynamic antibody response during persistent infection represents a significant gap in knowledge. A leading hypothesis is that persistent infection repeatedly stimulates short-lived plasma cells, which predominately remain in the spleen and have a 3- to 4-day half-life prior to undergoing apoptosis, rather than long-lived plasma cells which traffic to the bone marrow (20). Short-lived antibody and failure to maintain immunity are also features of Plasmodium sp. infection (33), which shares the common features of antigenic variation and persistence within the bloodstream. The requirements for transition to long-lived plasma cells are not completely understood. However, antigen-specific CD4⁺ T lymphocytes clearly provide some of the signals for this transition. Consistent with this hypothesis is the observation with A. marginale that, although robust CD4⁺ T-cell responses specific to both the HVR and conserved domains can be induced by MSP2 immunization (1), these cannot be consistently detected during actual infection (2). Dissecting the mechanism underlying the short-lived antibody responses, both from a structural basis of the HVR and the immunologic basis, will further enhance our understanding of how highly antigenically variant pathogens persist within the infected host. Furthermore, development of new immunization strategies that induce long-lived antibody responses may hold new promise for vaccine development against the numerous pathogens that use this mechanism of persistence.

 
This research was supported by NIH R01 AI44005 and USDA-ARS-CRIS 5348-32000-016-00D.

The statistical assistance of Marc Evans and assistance with MHC haplotyping by Junzo Norimine is gratefully acknowledged, as is the technical assistance of James Allison, Ralph Horn, and Bev Hunter.

 
* Corresponding author. Mailing address: 402 Bustad Hall, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6033. Fax: (509) 335-8529. E-mail: gpalmer{at}vetmed.wsu.edu

Published ahead of print on 4 September 2007.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: R. P. Morrison

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Infection and Immunity, November 2007, p. 5185-5190, Vol. 75, No. 11
0019-9567/07/$08.00+0     doi:10.1128/IAI.00913-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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