Microbial Immunity and Vaccines

Conformational Dependence ofAnaplasma marginale Major Surface Protein 5 Surface-Exposed B-Cell Epitopes

Devere Munodzana, Terry F. McElwain, Donald P. Knowles, Guy H. Palmer
DOI: 10.1128/IAI.66.6.2619-2624.1998

ABSTRACT

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denaturedA. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

Anaplasma marginale is an arthropod-borne ehrlichial pathogen of cattle that invades and replicates in mature erythrocytes (7). Acute infection is characterized by high levels of rickettsemia (>109 infected erythrocytes/ml) and severe anemia, which frequently results in abortion or death (5, 7). Immunity against acute A. marginale rickettsemia is directed against outer membrane surface proteins, and infectivity can be neutralized with antibodies against surface exposed epitopes (18, 20, 21). Correspondingly, cattle immunized with A. marginale outer membranes develop significantly lower rickettsemia following challenge than do adjuvant-immunized controls (20, 22, 27). Sera from these immunized and protected cattle recognize six major surface proteins (MSPs), and antibody titers against MSP2 and MSP5 correlate with protection against challenge with the homologous strain (20, 22, 27). In contrast to protection induced by immunization with whole outer membranes or a native MSP1a/MSP1b complex, isolated recombinant-expressed MSPs, either alone or in combination, fail to induce comparable protection against rickettsemia (17, 18, 20, 27). Consequently, we have hypothesized that MSP conformation, as determined by secondary through quatenary structures, is a critical determinant in the efficacy of experimental vaccines (13, 20, 30).

The outer membrane is composed of MSPs linked both covalently and noncovalently in multimeric complexes (30). MSP5 and MSP2 occur in both monomeric intramolecularly disulfide-bonded and multimeric intermolecularly disulfide-bonded forms in the membrane: MSP5 as a dimer and MSP2 as a tetramer (19, 30, 31). Importantly, both MSP5 and MSP2 bear immunodominant B-cell epitopes and, in outer membrane-immunized cattle, the antibody titer correlates with protection against challenge with the homologous A. marginale strain (19, 27, 31). Based on our hypothesis, we would predict that the MSP2 and MSP5 immunodominant surface-exposed epitopes are conformationally dependent and require disulfide bonding to maintain epitope conformation. We chose to first test this prediction with intramolecularly disulfide bonded MSP5. MSP5, in contrast to the antigenically variable MSP2 (2, 19), is encoded by a single highly conserved gene and expresses invariant surface epitopes recognized by outer membrane-immunized as well as previously infected immune cattle (1, 6, 14). In this paper, we report the disulfide bond and conformational requirements of defined MSP5 surface-exposed epitopes and the results of testing whether antibody binding to the A. marginale surface requires maintenance of secondary and tertiary structures.

MATERIALS AND METHODS

Physical mapping.ANAF16C1 is an immunoglobulin G1 (IgG1) monoclonal antibody (MAb) directed against the A. marginalesurface and binds MSP5 in all strains of A. marginale,A. ovis, and A. centrale tested (1, 6, 12, 14). Escherichia coli transformed with plasmid pAM104A expresses a full-length MSP5 polypeptide that is bound by MAb ANAF16C1 (31). Full-length and truncated msp5 clones expressed as fusion partners with maltose binding protein (MBP) were used to identify the MSP5 region bound by MAb ANAF16C1. Briefly, the entire msp5 open reading frame (nucleotides 118 to 753 based on the numbering of the original clone in pAM104A [31]) was amplified with forward and reverse primers incorporating XbaI recognition sites, digested, and ligated in frame into the XbaI site of the vector pMal-c2 (24). The plasmid encoding the full-length MSP5-MBP fusion was designated msp5.0, and the expressed protein was designated MSP5.0. The following truncated msp5 clones were generated by the same strategy with site-specific forward and reverse primers: msp5.1, a 371-bp clone representing bp 118 to 488;msp5.2, a 356-bp clone representing bp 390 to 745; andmsp5.3, a 483-bp clone representing bp 118 to 600. The sequences of all clones were verified by double-strand sequencing by primer extension with dideoxy chain termination (25).E. coli XL-1 Blue was transformed with each plasmid, and the expression of an MSP5-MBP fusion protein of the appropriate size was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant E. coli lysate and immunoblotting with detection by rabbit anti-MBP polyclonal antibody (3). The orientations of the full-length and truncated msp5constructs and the encoded proteins relative to the predicted conformation of native MSP5 are shown in Fig.1. Each MSP5-MBP fusion protein was purified on individual amylose affinity columns following extraction as soluble proteins from recombinant E. coli (24). Briefly, 2 × 108 bacteria per ml of rich medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.2% glucose), containing 100 μg of ampicillin per ml, was incubated in the same medium with the addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h at 37°C to induce fusion protein expression. Bacteria were disrupted by freezing and rapid thawing followed by sonication. The recombinant expressed proteins were collected in the supernatant and loaded on amylose columns with a binding capacity of 3 mg of MBP per ml of resin. The columns were washed and the recombinant fusion proteins were eluted as previously described (24). Eluted recombinant proteins were detected by immunoblotting with rabbit anti-MBP polyclonal antibody and then tested for reactivity with MAb ANAF16C1 by SDS-PAGE and immunoblotting (3). Antibody binding was detected by using horseradish peroxidase-labeled goat anti-rabbit IgG (for anti-MBP antibody) or goat anti-murine IgG (for MAbs) and enhanced chemiluminescence (3). Purified nonfusion MBP, and unrelated MBP fusion protein (MBP–Babesia bovis RAP-1 [26]), and uninfected erythrocytes were used as negative control antigens. A. marginale-infected erythrocytes and E. coli transformed with plasmid pAM104A were used as positive antigen controls (31). Normal rabbit serum and the IgG1 MAb Tryp1E1 were used as negative antibody controls.

Fig. 1.
Fig. 1.

Physical map of the recombinant MSP5 proteins relative to a transmembrane helical protein hydrophobicity-hydrophilicity profile of native MSP5. The map was generated with the Genetics Computer Group package from the University of Wisconsin. They axis reflects the Goldman-Engelman-Steitz hydrophobicity scale over a window of 20 residues, and the x axis represents the amino acid position in MSP5. The proteins expressed by full-length (msp-5.0) or truncated (msp-5.1,msp-5.2, and msp-5.3) recombinant clones are plotted against the same x axis, and the positions of the two cysteines are indicated by the letter C.

Conformational sensitivity of MAb ANAF16C1 binding.Affinity-purified MSP5.0 was incubated, at 10 μg per treatment (in duplicate), with either 8 M urea, 60 mM dithiothreitol (DTT), or 300 mM iodoacetamide (IA), or one of the combinations DTT and IA; urea and IA; or urea, DTT, and IA. The urea and DTT treatments were performed at 56°C for 12 h, and the IA treatment was performed for 1 h at 25°C (4). An untreated sample was incubated identically and used as a positive control. Reactivity was determined by immunoblotting (3) with MAb ANAF16C1 or the negative control MAb Tryp1E1.

Cattle previously immunized with purified A. marginale outer membranes developed high titers of anti-MSP5 antibody and were shown to be protected against acute rickettsemia upon challenge (27, 31). Serum obtained postimmunization but prechallenge was adsorbed with either denatured and reduced (8 M urea, 60 mM DTT, 300 mM IA) or untreated, native Norton strain organisms (28). As controls, serum either was left unadsorbed or was adsorbed by the identical method with either denatured and reducedE. coli or untreated E. coli. Adsorption, performed at 25°C for 1 h, was repeated until there was no reactivity with the adsorbing antigen preparation as determined by immunoblotting. Each serum treatment was then tested for inhibition of MAb ANAF16C1 binding to recombinant MSP5.0 by a competitive inhibition enzyme-linked immunosorbent assay (ELISA) as described previously (1, 6). Briefly, individual wells in 96-well plates were coated with 1 μg of amylose-resin-purified MSP5.0 fusion protein in 100 μl of carbonate-bicarbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3 [pH 9.6]). The wells were incubated for 1 h at room temperature with 200 μl of blocking buffer (250 mM K2HPO4, 250 mM KH2PO4, 0.5% fraction V bovine serum albumin, 0.75% glycine, 1% sucrose) and then washed four times with phosphate-buffered saline (PBS; pH 7.2). The adsorbed and unadsorbed test sera were diluted in PBS–1% BSA, to a final dilution of 1:40,000, the dilution of unadsorbed serum that resulted in approximately 70% inhibition of MAb ANAF16C1 binding to MSP5.0. Adsorbed, diluted sera were added to triplicate wells in 100-μl aliquots, and the wells were incubated at room temperature for 1 h. The wells were washed four times with 200 μl of PBS per well and then incubated for 15 min at room temperature with horseradish peroxidase-conjugated MAb ANAF16C1 as described previously (1, 6). After four additional washes with PBS, 50 μl of 0.5-μg/μl o-phenylenediamine hydrochloride dihydrochloride in substrate buffer (0.2 M Na2HPO4, 0.1 M citric acid) was added to each well. The plates were incubated for 10 min, and the reactions were terminated with 25 μl of 2 N H2SO4. The results were expressed as percent inhibition (and standard deviation) of MAb ANAF16C1 binding to MSP5.0 (1, 6).

Conformational dependence of antibody binding to A. marginale MSP5 surface exposed epitopes.Calves were obtained at 1 day of age and raised in a tick- and fly-free facility at the Central Veterinary Laboratory, Harare, Zimbabwe. Before immunization, sera were shown to be unreactive with A. marginale by immunoblotting against whole-organism lysate (11) and by the competitive inhibition MSP-5.0 ELISA (1, 6). Five calves were immunized by subcutaneous inoculation of 50 μg of native MSP5, purified from A. marginale on a MAb ANAF16C1 affinity column as described previously (31), in saponin adjuvant. The immunization was repeated three times at 3- to 4-week intervals. Five adjuvant control calves were given saponin alone by using the identical schedule and route of inoculation. Sera were obtained 1 month after the last inoculation, and the anti-MSP5 titer was determined by the competitive inhibition ELISA. As described in Results, all sera from MSP5-immunized calves had high titers of anti-MSP5 antibody. Two of these sera were then adsorbed with amylose resin-purified MSP5.0 or denatured and reduced (8 M urea, 60 mM DTT, 300 mM IA) purified MSP5.0. As controls, these sera either were left unadsorbed or were adsorbed, by using the identical protocol, with either denatured and reduced MBP or untreated MBP. Adsorptions, performed at 25°C for 1 h, were repeated until there was no reactivity with the adsorbing antigen preparation as determined by immunoblotting. Unadsorbed and adsorbed sera were then tested for binding to native surface exposed MSP5 epitopes by agglutination of purified A. marginale as previously described (19).

RESULTS

Physical mapping.The physical maps of the full-length and truncated MSP5-MBP fusion proteins expressed in pMal-c2 are shown in Fig. 1. Each fusion protein was purified on individual amylose affinity columns and identified by SDS-PAGE and immunoblotting with detection by rabbit polyclonal antibody specific for the MBP fusion partner. Figure2 shows the binding of anti-MBP antibody to the expected 65-kDa MSP5.0 fusion protein (lane 1) and to MBP alone (lane 2). The anti-MBP antibody also reacted with the truncated fusion proteins MSP5.1, MSP5.2, and MSP5.3 (data not shown) but not with purified A. marginale (lane 3). There was no binding of control normal rabbit sera to any of the MSP5-MBP fusion proteins (MSP5.0 is shown in lane 4), MBP (lane 5), or A. marginale (lane 6).

Fig. 2.
Fig. 2.

Expression of recombinant MSP5.0. Lanes 1 and 4 contain MSP5.0 fused to MBP and purified on an amylose affinity column; lanes 2 and 5 contain MBP alone; and lanes 3 and 6 contain a lysate ofA. marginale-infected erythrocytes. Lanes 1 to 3 were reacted with rabbit antiserum against MBP; lanes 4 to 6 were reacted with the same dilution of normal rabbit serum. The positions of the MSP5.0-MBP fusion protein and MBP alone are indicated in the left margin.

Each recombinant MSP5 fusion protein was then tested for reactivity with MAb ANAF16C1 or the Tryp1E1-negative control MAb by immunoblotting. MSP5.0 was bound by MAb ANAF16C1 (Fig.3, lanes 2 and 3) but not by an isotype control MAb, Tryp1E1 (lanes 6 and 7). This indicates that the presence of the MBP fusion partner does not alter recognition of the MSP5 epitope by MAb ANAF16C1. This MAb also bound A. marginale native MSP5 (lane 4). MAb ANAF16C1 did not react with the negative control B. bovis RAP-1–MBP fusion protein (lane 1). Of the truncated fusion proteins, only MSP5.3 was bound by MAb ANAF16C1 (Fig. 4, lane 4). ANAF16C1 did not bind MSP5.1 (lane 2), MSP5.2 (lane 3), or the negative controlB. bovis RAP-1–MBP fusion protein (lane 1). This reactivity indicates that not only is the amino-terminal region (nucleotides 118 to 390, encoding the first 91 amino acids including the conserved cysteine residues) necessary for ANAF16C1 binding but that also some or all of the region composed of amino acids 125 to 161 (encoded by nucleotides 492 to 600) is also required. These data, without further mapping, are consistent with conformational dependence of the immunodominant epitope bound by MAb ANAF16C1. Nonfusion MSP5 expressed by E. coli containing plasmid p104A was used as a positive control and was bound, as expected, by MAb ANAF16C1 (Fig. 4, lane 5).

Fig. 3.
Fig. 3.

MAb ANAF16C1 binds recombinant MSP5.0. Lanes 1 and 5 contain B. bovis RAP-1–MBP fusion protein as a negative antigen control; lanes 2, 3, 6, and 7 contain MSP5.0-MBP fusion protein (lanes 2 and 6 contain protein from a different column fraction from the protein in lanes 3 and 7); lanes 4 and 8 contain a lysate ofA. marginale-infected erythrocytes. Lanes 1 to 4 were reacted with MAb ANAF16C1; lanes 5 to 8 were reacted with the isotype control MAb Tryp1E1. The positions of the MSP5.0-MBP fusion protein and the native MSP5 are indicated in the left margin.

Fig. 4.
Fig. 4.

MAb ANAF16C1 binds recombinant MSP5.3 but not MSP5.1 or MSP5.2. Lanes 1 and 6 contain B. bovis RAP-1–MBP fusion protein as a negative antigen control; lanes 2 and 7 contain MSP5.1-MBP fusion protein; lanes 3 and 8 contain MSP5.2-MBP fusion protein; lanes 4 and 9 contain MSP5.3-MBP fusion protein; and lanes 5 and 10 contain nonfusion MSP5 expressed by E. coli containing plasmid p104A (31). Lanes 1 to 5 were reacted with MAb ANAF16C1; lanes 6 to 10 were reacted with the isotype control MAb Tryp1E1. The positions of the MSP5.3-MBP fusion protein and nonfusion MSP5 are indicated in the left margin.

Conformational sensitivity of MAb ANAF16C1 binding.The conformational dependence of MSP5 was tested by treatment of purified MSP5.0 with denaturing and reducing agents followed by determination of MAb ANAF16C1 binding. Reduction of disulfide bonds with DTT followed by covalent modification of sulfhydryl groups with IA to prevent reoxidation completely abolished MAb binding (Fig.5). This effect was probably due to the effect on disulfide bonding, since neither DTT nor IA alone had any detectable effect on the epitope (Fig. 5). This dependence on disulfide bonding is consistent with the epitope-mapping results, which showed a requirement for the amino-terminal half of MSP5, containing the conserved cysteine residues. Treatment with 8 M urea, which denatures the protein secondary structure, resulted in a partial loss of MAb ANAF16C1 binding (Fig. 5). This effect is again consistent with a conformationally dependent epitope and may involve both the amino- and carboxy-terminal hydrophilic regions.

Fig. 5.
Fig. 5.

MAb ANAF16C1 binding is sensitive to denaturation and reduction of MSP5. Purified MSP5.0 was either untreated or treated with 8 M urea (MSP5.0+urea); 60 mM dithiothreitol (MSP5.0+DTT); DTT and 300 mM IA (MSP5.0+DTT+IA); urea, DTT, and IA (MSP5.0+urea+DTT+IA); urea and IA (MSP5.0+urea+IA); or IA alone (MSP5.0+IA). Samples were reacted in duplicate with either MAb ANAF16C1 (left) or the negative control MAb Tryp1E1 (right).

To test whether recognition of the MSP5 immunodominant epitope by antibody from outer membrane-immunized and protected cattle was also conformationally dependent, serum was adsorbed with native or reduced and denatured A. marginale lysate and then tested for the ability to inhibit MAb ANAF16C1 binding. Unadsorbed serum was diluted (1:40,000) to achieve 70% inhibition of MAb ANAF16C1 binding (Table1). All test samples following adsorption were then tested at a final dilution of 1:40,000. As shown in Table 1, adsorption with native A. marginale significantly depleted bovine serum antibody inhibition of MAb ANAF16C1 binding. In contrast, adsorption with native E. coli or reduced and denatured A. marginale or E. coli did not significantly reduce the binding of the immune bovine serum to the MSP5 immunodominant epitope (Table 1). This indicates that the antibody response to this MSP5 epitope following effective outer membrane immunization is conformationally restricted.

View this table:
Table 1.

Binding of antibody induced by outer membrane immunization to the MSP5 immunodominant epitope is dependent on native conformation of A. marginale

Conformational dependence of antibody binding to A. marginale MSP5 surface-exposed epitopes.Immunization of cattle with native MSP5 induced high titers of antibody against the immunodominant MSP5 epitope, as determined by the competitive inhibition ELISA based on ANAF16C1 binding (data not shown). Sera from two of the MSP5-immunized cattle were then used to determine if recognition of MSP5 epitopes on the A. marginalesurface was conformationally dependent. Unadsorbed sera had end-point agglutination titers of 512, while adsorption with native MSP5.0 diminished surface binding 32- and 64-fold, respectively, for each of the two test sera (Table 2). In contrast, adsorption with denatured and reduced MSP5.0 lysate either did not alter (anti-MSP5.0 serum 1) or only slightly diminished (anti-MSP5.0 serum 2) surface reactivity compared to negative control adsorptions with either native or reduced and denatured MBP (Table 2). Sera from the five cattle immunized with saponin alone had end-point agglutination titers of 4 or less (data not shown). These results indicate that the antibody response to MSP5, as presented on theA. marginale surface, is predominantly against conformationally dependent epitopes.

View this table:
Table 2.

Binding of anti-MSP5 sera to the A. marginale surface requires reactivity with native, nondenatured epitopes

DISCUSSION

Why individual MSPs fail to induce protection at a level comparable to that induced by immunization with intact A. marginale outer membranes is unknown and represents an important gap in our knowledge needed to develop and improve vaccines against ehrlichial pathogens. Possible explanations, which are not mutually exclusive, include the following: (i) each MSP alone induces partially protective immunity, and the efficacy of the outer membrane complex simply reflects the sum of the individual components; (ii) the multimeric outer membrane complex enhances antigen presentation compared to soluble individual MSPs and generates a phenotypically different immune response; and (iii) induction of protection requires antibody to conformationally dependent epitopes on theA. marginale surface. The first possibility is not congruent with data showing that combinations of up to three MSPs do not consistently enhance protection compared to that afforded by immunization with individual MSPs (17, 18, 20, 23). In contrast, both the second and third explanations remain viable. As an entrée to investigating the importance of antibody against conformationally dependent epitopes, we analyzed the structural requirements of a highly conserved immunodominant epitope on MSP5. This epitope, defined by binding of MAb ANAF16C1, is conserved among all tested strains of A. marginale, A. centrale, and A. ovis and induces high titers of antibody in all infected species including cattle, sheep, and goats (1, 6, 14, 31). Initial physical mapping of the epitope with full-length and truncated recombinant expressed MSP5 indicated that residues encoded 5′ to nucleotide 390 (amino acid 91) as well as some or all of the region encoded by nucleotides 492 to 600 (amino acids 125 to 161) were required. Importantly, the required amino-terminal region included the conserved cysteines (31), consistent with the proposed importance of intramolecular disulfide bonding in the MSP conformation (30). The absolute dependence of the immunodominant epitope on disulfide bonding was confirmed by the loss of MAb ANAF16C1 binding to MSP5.0 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. Interestingly, MAb binding was also reduced after urea treatment alone (Fig. 5). This suggests that secondary protein structure, apart from the tertiary requirements for intramolecular disulfide bonding, is also needed for epitope conformation, a finding consistent with the physical mapping results indicating contributions from two distant hydrophilic regions of MSP5. Whether amino acids in these disparate regions are juxtaposed to form the actual epitope (defined by binding to the complementarity determining regions of the antibody) or whether the epitope is encoded within one of the regions and the second is required only to provide correct secondary structure for binding is unknown. Both scenarios are consistent with the requirement for disulfide bonding in or adjacent to a hydrophobic segment interposed between two hydrophilic and presumed surface-exposed regions of MSP5 (Fig. 1).

The single MSP5 epitope defined by MAb ANAF16C1 binding was analyzed as a model for immunodominant MSP epitopes (11, 20, 30). The presence of conserved cysteines and disulfide bonds in MSP2 and MSP4 (15, 19, 30) suggests that conformational dependence may be a common feature among A. marginaleMSPs. In addition, the presence of an MSP5 homolog in Cowdria ruminantium MAP-2 (8) and of MSP2 homologs inC. ruminantium MAP1 (19, 29) and Ehrlichia chaffeenesis OMP-1 (16) provides support for broad applicability of this model among ehrlichial pathogens.

Importantly, antibodies induced by outer membrane immunization, which results in high anti-MSP5 antibody titers that correlate with protection against homologous challenge (27), recognized the MSP5 immunodominant epitope in a conformationally dependent form, as shown by the results in Table 1. Furthermore, the polyclonal antibody induced by native MSP5 immunization also recognized predominantly conformationally dependent epitopes on theA. marginale surface. This indicates that the surface binding of antibody to all MSP5 immunodominant epitopes is conformationally dependent and is consistent with a requirement for native-protein secondary and tertiary structures in effective immunization.

In contrast to the secondary- and tertiary-structure requirements for MSP5 B-cell epitopes, the role of the quatenary structure remains unclear. Membrane MSP5 and MSP2 occur as both intramolecularly disulfide bonded monomers and intermolecularly disulfide linked multimers. Although monomeric MSPs, including MSP2 and MSP5, induce antibody against the A. marginale surface, complete neutralization of infectivity may require antibody directed against functional surface regions composed of two or more MSPs (13, 30). This possibility is suggested by the greater inhibition ofA. marginale binding to the erythrocyte surface by antibodies generated against native organisms or a complex of MSP1a and MSP1b compared to antibody generated against MSP1a and MSP1b individually (9, 10). The importance of antibody against multiple MSPs is also supported by the complete neutralization of in vivo infectivity by antibody generated against the intact outer membranes (21). Whether intermolecular bonding of MSPs results in different B-cell epitopes from those resulting from intramolecularly bonded MSPs is unknown, although the high degree of conformational dependence shown in the present study suggests that changes in bonding pattern are likely to alter the surface-exposed epitopes. Consequently, defining the structural requirements of critical outer membrane epitopes is a priority and will support the development and testing of vaccines that maintain native MSP structure. These approaches include recombinant MSP immune system-stimulating complexes, expression of multiple recombinant MSPs in the outer membranes of live bacterial vectors, and direct immunization with DNA encoding MSPs.

ACKNOWLEDGMENTS

This work was supported by U.S. Agency for International Development grant 263-0152-A-00-2207-00, The Central Veterinary Laboratory of Zimbabwe, U.S. Department of Agriculture National Research Initiative Competitive Grants Program grant 95-37204-2348, and USDA-BARD grant US-2238-92C.

We acknowledge Beverly Hunter, Carla Robertson, and Will Harwood for technical assistance and Unesu Ushewokunze-Obatolu for her continuous support of this project and of D.M.

FOOTNOTES

    • Received 30 December 1997.
    • Returned for modification 4 March 1998.
    • Accepted 18 March 1998.

REFERENCES

  1. 1.↵
    1. Echaide S. T.,
    2. Knowles D. P.,
    3. McGuire T. C.,
    4. Palmer G. H.,
    5. Suarez C. E.,
    6. McElwain T. F.
    Detection of cattle naturally infected with Anaplasma marginale in an endemic region using nested PCR and recombinant MSP5-cELISA. J. Clin. Microbiol. 36 1998 777 782
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Eid G.,
    2. French D. M.,
    3. Lundgren A.,
    4. Barbet A. F.,
    5. McElwain T. F.,
    6. Palmer G. H.
    Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia. Infect. Immun. 64 1996 836 841
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Gallagher S.,
    2. Winston S. E.,
    3. Fuller S. A.,
    4. Hurrell J. G. R.
    Immunoblotting and Immunodetection Current protocols in molecular biology Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 2 1993 10.8.1 10.8.17 John Wiley & Sons, Inc. New York
    OpenUrl
  4. 4.↵
    1. Hotzel I.,
    2. Brown W. C.,
    3. McElwain T. F.,
    4. Rodriguez S. D.,
    5. Palmer G. H.
    Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1 (RAP-1). Mol. Biochem. Prasitol. 81 1996 89 99
    OpenUrl
  5. 5.↵
    1. Kieser S. T.,
    2. Eriks I. S.,
    3. Palmer G. H.
    Cyclic rickettsemia during persistent Anaplasma marginale infection of cattle. Infect. Immun. 58 1990 1117 1119
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Knowles D. P.,
    2. Torioni de Echaide S.,
    3. Palmer G. H.,
    4. McGuire T. C.,
    5. Stiller D.,
    6. McElwain T. F.
    Antibody against an Anaplasma marginale MSP-5 epitope common to tick and erythrocyte stages identifies persistently infected cattle. J. Clin. Microbiol. 34 1996 2225 2230
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Losos G. J.
    Anaplasmosis Infectious tropical diseases of domestic animals. Losos G. J. 1986 742 795 Longman Press Harlow, United Kingdom
  8. 8.↵
    1. Mahan S. M.,
    2. McGuire T. C.,
    3. Semu S. M.,
    4. Bowie M. V.,
    5. Jongejan F.,
    6. Rurangirwa F. R.,
    7. Barbet A. F.
    Molecular cloning of a gene encoding the immunogenic 21-kDa protein of Cowdria ruminantium. Microbiology 140 1994 2135 2142
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. McGarey D. J.,
    2. Allred D. R.
    Characterization of hemagglutinating components of the Anaplasma marginale initial body surface and identification of possible adhesins. Infect. Immun. 62 1994 4587 4593
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. McGarey D. J.,
    2. Barbet A. F.,
    3. Palmer G. H.,
    4. McGuire T. C.,
    5. Allred D. R.
    Putative adhesins of Anaplasma marginale major surface polypeptides (MSP) 1a and 1b. Infect. Immun. 62 1994 4594 4601
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. McGuire T. C.,
    2. Davis W. C.,
    3. Brassfield A. L.,
    4. McElwain T. F.,
    5. Palmer G. H.
    Identification of Anaplasma marginale long-term carrier cattle by detection of serum antibody to isolated MSP-3. J. Clin. Microbiol. 29 1991 788 793
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. McGuire T. C.,
    2. Palmer G. H.,
    3. Goff W. L.,
    4. Johnson M. I.,
    5. Davis W. C.
    Common and isolate restricted antigens of Anaplasma marginale detected with monoclonal antibodies. Infect. Immun. 45 1984 697 700
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Musoke A. J.,
    2. Palmer G. H.,
    3. McElwain T. F.,
    4. Nene V.,
    5. McKeever D.
    Prospects for subunit vaccines against tick-borne diseases. Br. Vet. J. 152 1996 621 639
    OpenUrlPubMed
  14. 14.↵
    1. Ndung’u L. W.,
    2. Aguirre C.,
    3. Rurangirwa R. R.,
    4. McElwain T. F.,
    5. McGuire T. C.,
    6. Knowles D. P.,
    7. Palmer G. H.
    Detection of Anaplasma ovis infection in goats using the MSP5 competitive inhibition ELISA. J. Clin. Microbiol. 33 1995 675 679
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Oberle S. M.,
    2. Barbet A. F.
    Derivation of the complete msp-4 gene sequence of Anaplasma marginale without cloning. Gene 136 1993 291 294
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Ohashi N.,
    2. Zhi N.,
    3. Zhang Y.,
    4. Rikihisa Y.
    Immunodominant major outer membrane proteins of Ehrlichia chaffeensis are encoded by a polymorphic multigene family. Infect. Immun. 66 1998 132 139
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Palmer G. H.,
    2. Barbet A. F.,
    3. Cantor G. H.,
    4. McGuire T. C.
    Immunization of cattle with the MSP-1 surface protein complex induces protection against a structurally variant Anaplasma marginale isolate. Infect. Immun. 57 1989 3666 3669
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Palmer G. H.,
    2. Barbet A. F.,
    3. Davis W. C.,
    4. McGuire T. C.
    Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science 231 1986 1299 1302
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Palmer G. H.,
    2. Eid G.,
    3. Barbet A. F.,
    4. McGuire T. C.,
    5. McElwain T. F.
    The immunoprotective Anaplasma marginale major surface protein-2 (MSP-2) is encoded by a polymorphic multigene family. Infect. Immun. 62 1994 3808 3816
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Palmer G. H.,
    2. McElwain T. F.
    Molecular basis for vaccine development against anaplasmosis and babesiosis. Vet. Parasitol. 57 1995 233 253
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Palmer G. H.,
    2. McGuire T. C.
    Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol. 133 1984 1010 1015
    OpenUrlAbstract
  22. 22.↵
    1. Palmer G. H.,
    2. Munodzana D.,
    3. Tebele N.,
    4. Ushe T.,
    5. McElwain T. F.
    Heterologous strain challenge of cattle immunized with Anaplasma marginale outer membranes. Vet. Immunol. Immunopathol. 42 1994 265 273
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Palmer G. H.,
    2. Oberle S. M.,
    3. Barbet A. F.,
    4. Davis W. C.,
    5. Goff W. L.,
    6. McGuire T. C.
    Immunization with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma marginale challenge. Infect. Immun. 56 1988 1526 1531
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Riggs P.
    Expression and purification of maltose-binding protein fusions Current protocols in molecular biology Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 2 1994 16.6.1 16.6.14 John Wiley & Sons, Inc. New York, N.Y
    OpenUrl
  25. 25.↵
    1. Slatko B. E.,
    2. Albright L. M.,
    3. Tabor S.
    DNA sequencing by the dideoxy method Current protocols in molecular biology Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1 1994 7.4.1 7.4.35 John Wiley & Sons, Inc. New York, N.Y
    OpenUrl
  26. 26.↵
    1. Suarez C. E.,
    2. Palmer G. H.,
    3. Jasmer D. P.,
    4. Hines S. A.,
    5. Perryman L. E.,
    6. McElwain T. F.
    Characterization of the gene encoding a 60kD Babesia bovis merozoite protein with conserved and surface exposed epitopes. Mol. Biochem. Parasitol. 46 1991 45 52
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Tebele N.,
    2. McGuire T. C.,
    3. Palmer G. H.
    Induction of protective immunity using Anaplasma marginale initial body membranes. Infect. Immun. 59 1991 3199 3204
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Tebele N.,
    2. Palmer G. H.
    Crossprotective immunity between the Florida and a Zimbabwe stock of Anaplasma marginale. Trop. Anim. Health Prod. 23 1991 197 202
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Van Vliet A. H. M.,
    2. Jongejan F.,
    3. Van Kleef M.,
    4. Van Der Zeist B. A. M.
    Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium. Infect. Immun. 62 1994 1451 1456
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Vidotto M.,
    2. McGuire T. C.,
    3. McElwain T. F.,
    4. Palmer G. H.,
    5. Knowles D. P.
    Intermolecular relationships of major surface proteins of Anaplasma marginale. Infect. Immun. 62 1994 2940 2946
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Visser E. S.,
    2. McGuire T. C.,
    3. Palmer G. H.,
    4. Davis W. C.,
    5. Shkap V.,
    6. Pipano E.,
    7. Knowles D. P.
    The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species. Infect. Immun. 60 1992 5139 5144
    OpenUrlAbstract/FREE Full Text
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KEYWORDS

Anaplasma
Antigens, Bacterial
B-Lymphocytes
Bacterial Outer Membrane Proteins
epitopes

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