Molecular basis for vaccine development against anaplasmosis and babesiosis

veterinary parasitology ELSEVIER

VeterinaryParasitology57 (1995) 233-253

Molecular basis for vaccine development against anaplasmosis and babesiosis G u y H. Palmer*, Terry F. M c E l w a i n Department of VeterinaryMicrobiologyand Pathology, WashingtonState University,Pullman, WA 99164, USA

Abstract

Immunization of livestock against the erythroparasitic pathogens Anaplasma marginale, Babesia bigemina, and Babesia boris with safe and effective killed vaccines is not yet feasible on a practical basis. However, the immune protection afforded by recovery from natural infection and premunition indicates that microbial epitopes capable of inducing immunity exist and that the bovine immune system can be primed appropriately. Induction of protection by immunization with killed parasite fractions, enriched for polypeptides with surface exposed epitopes, supports a focus on surface epitopes, including apical complex organellar epitopes in Babesia, for vaccine development. Cloning, sequencing, and expression of genes encoding these key surface polypeptides has allowed examination of polypeptide function and detailed analysis of epitope conservation in light of genetic polymorphism. In this paper, the characterization of these polypeptides at the epitope level and their roles in inducing protective immunity are reviewed. Definition of these epitopes, in combination with improved understanding of immune mechanisms, provides the basis for development of effective recombinant vaccines against anaplasmosis and babesiosis.

Keywords:Anaplasma marginale;Babesia spp.; Vaccines

1. Introduction

The major arthropod-borne hemoparasitic diseases of cattle--anaplasmosis, babesiosis, cowdriosis, theileriosis, and trypanosomiasis---are widespread throughout tropical and subtropical regions worldwide (Pino, 1981 ). The most prevalent of these diseases are anaplasmosis, caused by Anaplasma marginale, and babesiosis due to Babesia bigemina or Babesia boris infection. Following * Correspondingauthor: Tel. ( 509 ) 335-6033. 0304-4017/95/$09.50 © 1995 ElsevierScienceB.V. All rightsreserved SSDI 0304-4017 ( 94 )03123-1

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transmission to cattle, both Anaplasma and Babesia invade and replicate in mature erythrocytes resulting in anemia which may culminate in marked weight loss, abortion in pregnant cows, or death (Wright, 1973; Ristic, 1977; reviewed by Purnell, 1981 ). Despite the severity of losses and the widespread distribution of infection, effective control of anaplasmosis and babesiosis has not been achieved on a sustainable basis in most affected areas (Pino, 1981 ). Available control measures include: (i) acaracide-based control of arthropod transmission; (ii) vaccination, including premunition, of susceptible cattle (reviewed in Losos, 1986a,b). Control of arthropod transmission by regular acaricide application is most effective for Babesia bigemina and Babesia boris, which are transmitted primarily by one-host Boophilus ticks (Losos, 1986b; Friedhoff, 1988). However, owing to the high cost of effective acaricides this method of control is not sustainable for many livestock producers. Dependence upon acaracide control creates a highly susceptible cattle population vulnerable to renewed transmission upon disruptions in the control program, development of acaricide resistance, and transmission by other means such as biting flies carryingA, marginale (Lawrence et al., 1980; Norval, 1983 ). In contrast, vaccination provides a means to efficiently and economically protect cattle both within and imported into enzootic regions. The induction of protective immunity following either natural infection with Anaplasma or Babesia or deliberate infection (premunition) with less virulent strains indicates that immunoprophylaxis is an achievable goal (Mahoney, 1973; reviewed by Ristic, 1977; Callow, 1977; Palmer, 1989 ). Premunition is the most common method of immunoprophylaxis in enzootic areas. However, premunition has a number of biological and technical shortcomings that have limited its acceptability and sustainability including: (i) potential contamination of the blood vaccine with known pathogens (bovine leukosis virus, bluetongue virus, Theileria, or heterologous Anaplasma or Babesia strains) (Callow, 1977; Rogers et al., 1988); (ii) potential contamination and widespread direct transmission of unidentified or newly emergent pathogens; (iii) variation in virulence resulting in morbidity in inoculated adult cattle (Callow, 1977; Potgeiter and Van Rensburg, 1983; Lohr, 1969; Henry et al., 1983); (iv) loss of infectivity resulting in failure to induce premunition; (v) induction of immune responses against host erythrocyte antigens (Dennis et al., 1970; Ristic et al., 1981 ); (vi) dependence upon liquid nitrogen storage and field transport (Callow, 1977 ). These shortcomings, which have prevented licensure of live blood based vaccines in the US, have stimulated research on developing an effective inactivated vaccine. Inactivated vaccines based on either intact Anaplasma and Babesia or organism fractions have been shown to protect cattle against severe morbidity and mortality upon challenge (Brock et al., 1965; Mahoney, 1967a; reviewed by Ristic and Montenegro-James, 1988). This efficacy, although not optimal, clearly demonstrates that development of effective inactivated vaccines for these diseases is achievable. Reliance on experimentally infected splenectomized cattle as donors for parasitized erythrocytes (frequently used for Babesia and required for Anaplasma vaccines as there is no continuous in vitro cultivation for Ana-

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plasma), difficulty in achieving acceptable purification from contaminating erythrocyte antigens, and subsequent standardization problems result in both a high cost of production and variable efficacy (reviewed by Goodger, 1989; Palmer, 1989). In vitro cultivation for Babesia has provided a valuable research tool (Levy and Ristic, 1980) but the requirement for continual addition of bovine erythrocytes and low number of organisms obtained severely limit the sustainability of culture for production of vaccine antigen. These specific problems with the Anaplasma and Babesia vaccines largely reflect problems associated with many whole bacterial or protozoal vaccines. Consequently, there is increased focus on development of immunization strategies based on defined epitopes presented to the immune system in a context that will induce protective immune responses. From a practical standpoint, a carefully designed multivalent vaccine for all of the major arthropod-borne diseases of cattle may be the only acceptable method of preventing these diseases in co-endemic areas. Such a multivalent vaccine poses constraints on antigenic composition that limits antigen selection only to relevant B and T cell epitopes. Therefore, definition of these epitopes is paramount to success. In this review, we will describe current research in definition of critical Anaplasma and Babesia epitopes and their relevance to induction of protective immunity.

2. Anaplasma marginale:. Definition of vaccine antigens Protective immunity against Anaplasma marginale develops following recovery from acute infection and can be induced by immunization with killed whole organism vaccines (Brock et al., 1965; Montenegro-James et al., 1991 ). This efficacy clearly indicates that epitopes capable of inducing protective immunity exist and that cattle can mount the appropriate immune responses. However, the epitopes responsible for inducing protective immunity following infection or killed whole organism immunization are unknown and are not easily identified within these complex immunogens. To focus on a subset of whole organism epitopes, we have hypothesized that epitopes present on the A. marginale outer membrane are capable of inducing protective immunity (Palmer and McGuire, 1984). Bacterial outer membrane polypeptides are surface exposed and are involved in critical functions such as nutrient transport and attachment and invasion of host cells. Importantly, although the mechanisms of immunity against A. marginale are incompletely defined, epitopes on these molecules are also exposed to antibody and cell mediated effector mechanisms. In this review, the identification and characterization of outer membrane polypeptide immunogens and their relevance to protective immunity will be presented. 3. Immunization with outer membrane polypeptides

To induce a polyclonal immune response against multiple surface polypeptides, we incorporated purified A. marginale outer membrane polypeptides, from

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the Norton Zimbabwe strain, in Quil A (Tebele et al., 1991 ). Immunization of cattle with the outer membrane-Quil A matrix induced immunity against challenge with live virulent first-passage Norton, Zimbabwe A. marginale (Tebele et al., 1991 ). Importantly the level of protection, complete prevention of detectable rickettsemia in a majority of vaccinates, was indistinguishable from the immunity induced by premunition (Tebele and Palmer, 1991 ). The successful induction of immunity similar to premunition supports a critical role for outer membrane epitopes in protective immunity. Characterization of the outer membrane fraction revealed the presence of at least six major polypeptides, designated Major Surface Proteins (MSP), including the initial description of MSP- 5 (Tebele et al., 1991 ). MSP- 1a, MSP- 1b, MSP2, MSP-3, and MSP-4 had been previously identified by surface radioiodination of intact A. marginale isolated from infected erythrocytes (Palmer and McGuire, 1984). Interestingly, protection in outer membrane immunized cattle correlates (r= 0.97, P < 0.05 ) with antibody titer to these polypeptides (Tebele et al., 1991 ). The localization of the MSPs to the outer membrane and the correlation between B cell immunogenicity and protection is consistent with one or more of the MSPs being critical to induction of protective immunity. To date, several of the MSPs have been tested for induction of protective immunity using either purified native or recombinant polypeptides. Immunization of cattle with native or recombinant MSP-1, native MSP-2, and native or recombinant MSP-4 has induced protection, defined as statistically significant reduction in peak rickettsemia and anemia compared with adjuvant immunized control cattle, against acute anaplasmosis (Palmer et al., 1986a, 1988a, 1989b; T.C. McGuire, unpublished data, 1992; A.F. Barbet, unpublished data, 1994). Although this efficacy supports the outer membrane polypeptide approach and indicates that subunit induced immunity is achievable, none of the immunogens have been rigorously optimized to determine if the level of protection is sufficient for protection in the field. The level of protection in the limited trials to date is not uniform among individual vaccinates and complete protection against rickettsemia and anemia is achieved in only a minority of vaccinates. The remaining vaccinates are either partially protected (develop a reduced rickettsemia compared with adjuvant-immunized controls, but still develop anemia as indicated by significant decreases in hematocrit) or unprotected. This observation, significant protectionon a group basis but variable on an individual basis, occurs following immunization with MSP-1, MSP-2, and MSP-4 (Palmer et al., 1986a, 1988a, 1989b; T.C. McGuire, unpublished data, 1992; A.F. Barbet, unpublished data, 1994 ). The dichotomy in the level and uniformity of protection induced by the outer membrane-Quil A immunogen versus the individual polypeptides may reflect either the need for a multicomponent immunogen to induce a broad B and T cell polyclonal response or, alternatively, differences in antigen presentation. Analysis of MSP intermolecular relationships in the membrane revealed that MSP-2 occurs as a tetramer and MSP-5 as a dimer (M.C. Vidotto et al., 1994). In addition, MSP-I a and MSP-lb are disulfide bonded and non-covalently linked to MSP-5. These intermolecular relationships may be critical in inducing the appro-

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priate immune responses and are not maintained by the use of purified recombinant polypeptides. Consequently, presentation of recombinant MSPs in a membrane context may be essential to mimic the efficacy of the outer membrane immunogen. Quil-A, used as the adjuvant and matrix for the immunoprotective A. marginale outer membranes, forms a micellar structure above the critical micellar concentration (Dalsgaard, 1978). This micellar matrix, composed of the adjuvant, membrane lipids, and the amphipathic membrane polypeptides, has been shown to enhance immunogenicity of microbial surface proteins via both slow release of antigen and localization into the spleen with specific enhancement of both B and helper T lymphocyte responses (Scott et al., 1985; Flebbe and Braley-Mullen, 1986). The induction of appropriate responses, perhaps via membrane presentation, is essential for development of recombinant vaccines. 4. Characterization of recombinant M S P s

Cloning and expression of genes encoding A. marginale MSPs has provided, as noted above, defined immunogens. In addition, sequences of the individual MSPs provide information and reagents for identification of critical functional regions, including B and T cell epitopes, and for analysis of genetic polymorphism both within and between strains. MSP-l, originally described in the Florida strain as AmF 105 (Palmer and McGuire, 1984 ), is composed of disulfide linked MSP- 1a and MSP-lb and remains the best characterized of the A. marginale MSPs. Recent data by McGarey et al. (1994) demonstrates that the MSP- 1 complex and both MSP-la and MSP-lb individually mediate adherence to bovine erythrocytes. This suggests that MSP-la and MSP-lb serve as adhesins and may be required for erythrocyte invasion. Purified native MSP-l complex induces protection against challenge with homologous and at least one heterologous strain (Palmer et al., 1986, 1989). In contrast to native MSP-1, immunization with either recombinant MSP-1 a or MSP-1 a/b provides inconsistent protection with only a minority being completely protected from disease (T.C. McGuire, unpublished data, 1992). Immunization with non-complexed MSP-Ib has not induced significant immunity. MSP-lb is encoded by a multigene family composed of a minimum of four distinct copies with genetic polymorphism among strains (Barbet and AUred, 1991 ). Several of the gene copies may be simultaneously expressed but which copies are incorporated into the native MSP-1 complex as it exists in the A. marginale membrane is unknown. Dissection of this intermolecular relationship between MSP-1 a and polymorphic MSP-lb may be necessary to optimize recombinant MSP-1 immunization. In contrast to MSP-lb, MSP-1 a is encoded by a single gene copy with striking size polymorphism among strains (Oberle et al., 1988; Allred et al., 1990). Despite the polymorphism, owing to variable numbers of tandem 28 or 29 amino acid repeats in the protein, a neutralization-sensitive B cell epitope, composed of six amino acids contained within the 29-mer, is highly conserved among strains with no negative strains identified to date (McGuire et al., 1984; Palmer et al., 1986a, 1988b; Allred et al., 1990). Importantly, this epitope is present on organisms within 100% of the infected

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erythrocytes (McGuire et al., 1984). Immunization of cattle with a polymer of the 29 amino acid repeat induced lymphocyte proliferation, indicative of a previously undefined T cell epitope, and antibodies against the native organism including the neutralization-sensitive B cell epitope (T.F. McElwain, unpublished data, 1990). If MSP-1 a is indeed required for erythrocyte invasion, as is suggested by its adherence to erythrocytes, this may explain the invariance of the neutralization-sensitive epitope among strains and its expression in A. marginale stages within the infected tick vector (Palmer et al., 1985 ). MSP-2, originally described as a 36 kDa protein in the Florida strain (Palmer and McGuire, 1984), is a relatively immunodominant surface protein with epitopes shared between A. centrale and A. marginale (Adams and Smith, 1988; Nakamura et al., 1991; Shkap et al., 1991 ). Immunization of cattle with native MSP-2 has been shown to induce protection against homologous or heterologous strain challenge (Palmer et al., 1988a). As msp-2 has only recently been cloned and expressed (Palmer et al., 1994), no recombinant immunization experiments have been done. Although a panel of monoclonal antibodies against MSP-2 bind A. centrale and all strains of A. marginale examined thus far, there is variable expression of MSP-2 epitopes during infection (McGuire et al., 1984; Palmer et al., 1988b). Individual MSP-2 epitopes, including surface epitopes, are expressed on only a subset, approximately 70%, of infected erythrocytes. This interesting observation indicates that regulation of msp-2 expression occurs during infection. The recent cloning, sequencing, and expression of msp-2 may provide some clues as to the mechanisms underlying this observation. The single msp-2 copy expressed to date reacts with only a subset of the anti-MSP-2 monoclonal antibodies (Palmer et al., 1994). However, restriction mapping has indicated the presence of multiple partially homologous msp-2 gene copies widely dispersed throughout the chromosome (Palmer et al., 1994). The existence of multiple polymorphic rasp-2 copies explains, at least in part, the observations that not all organisms within a strain are reactive with a given anti-MSP-2 monoclonal antibody (McGuire et al., 1984). It may be that multiple rasp-2 copies are expressed in a regulated manner during erythrocyte invasion and subsequent replication. Alternatively, there may be multiple clones within a strain that express both unique and common MSP-2 epitopes. Consistent with previously published figures showing multiple MSP-2 polypeptides in purified native MSP-2 (Palmer et al., 1985 ), recent data indicates that a single organism may co-express different msp2 copies (Palmer et al., 1994). The biological significance and relevance for immunization of the large percentage of the A. marginale genome devoted to encoding MSP-2 and the striking MSP-2 polymorphism are yet unknown. Msp-2 is also expressed in tick stages ofA. marginale (Palmer et al., 1985 ) raising the possibility that different msp-2 copies may be involved in growth and replication within the arthropod vector. Interestingly, MSP-2 shares significant nucleotide and amino acid similarity to the single copy MSP-4 and the 32 kDa Cowdria ruminantium MAP- 1 (Palmer et al., 1994). Far less striking but significant similarity is shared

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with Merozoite Surface Antigen- 1 of Plasmodium falciparum suggesting the possibility of a common mechanism in erythrocyte invasion or replication. MSP-3 has been defined as an 86 kDa outer membrane polypeptide that is highly immunodominant during either experimental or natural infection (Palmer et al., 1986b; McGuire et al., 1991 ). Sera from cattle infected with different A. marginale strains all recognize MSP-3 in the Florida strain, indicating that epitopes may be broadly conserved (McGuire et al., 1991 ). MSP-3, similar to MSP-2, has epitopes conserved between A. centrale and A. marginale (Shkap et al., 1991 ). Cattle immunized with live A. centrale develop high titers of antibody against both MSP-2 and MSP-3. Whether this reflects a role in the cross-species immunity induced by A. centrale is unknown. In contrast, MSP-3 does not appear to be immunodominant in cattle immunized and protected by outer membrane immunization (Tebele et al., 1991 ). Cattle immunized with native purified MSP3 had a delayed onset of rickettsemia, as compared to adjuvant immunized control cattle, but were not statistically different in peak rickettsemia or severity of anemia (T.C. McGuire, unpublished data, 1992). At the molecular level, MSP-3 is not currently well characterized but appears to be encoded by a multigene family (G.H. Palmer, unpublished data, 1992; A.R. AUeman and A.F. Barbet, unpublished data, 1994). Characterization of the genes encoding MSP-3 will allow identification of immunodominant epitopes shared among strains and contribute to our understanding of the genetic regulation of polymorphic MSPs. MSP-4 and MSP-5 are recently characterized outer membrane proteins encoded by single copy genes (Visser et al., 1992; Obede et al., 1993 ). Both msp-4 and msp-5 express epitopes conserved among widely divergent strains and with A. centrale. In addition, rasp-5 is also expressed in A. ovis and in salivary gland stages ofA. marginale (Visser et al., 1992; D.P. Knowles, unpublished data, 1993 ). The functions of MSP-4 and MSP-5 are currently unknown. Although cattle immunized with either native or recombinant MSP-5 were not protected from challenge, compared with adjuvant-immunized control cattle, both native and recombinant MSP-4 induced protection against homologous challenge (A.F. Barbet,unpublished data, 1994).

5. Mechanisms of immunity induced by MSPs The statistically significant protection induced by immunization with native or recombinant MSPs is promising for development of improved disease control. However vaccines based on any of the recombinant MSPs would require substantial improvement prior to deployment. Currently, owing to a lack of knowledge regarding the protective immune mechanisms, optimization is done purely on an empirical basis by immunization and challenge. Knowledge of the required immune mechanisms would allow specific targeting of these mechanisms and more complete analysis of the responsiveness of individual vaccinates. Unfortunately, very little research on immune mechanisms in anaplasmosis has been reported in the last decade and our current understanding lags far behind what is known for other hemoparasitic infections. Studies on cattle persistently infected with A. rnarginale, which are solidly pro-

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tected against a subsequent challenge, suggest that cell-mediated mechanisms are critical in post-infection immunity. Immunity correlated with both inhibition of leukocyte migration and development of cutaneous hypersensitivity (Buening, 1976; Carson et al., 1976). While neither assay pinpoints a specific mechanism or a causal relationship, these results are consistent with a role for macrophage activation and specific T lymphocyte responsiveness. The involvement of specific antibody with these mechanisms, such as enhanced opsonization and phagocytosis by activated macrophages, has not been described in post-infection immunity. Antibody as a sole effector of post-infection immunity does not appear likely as passive transfer of antibodies from immune cattle, using the same basic procedure shown to transfer immunity against Babesia boris, did not protect recipient calves from challenge (Gale et al., 1992). Similarly, splenectomy or chemical immunosuppression with either dexamethasone or cyclophosphamide results in recrudescence in as few as 10 days, a period in which IgG antibody would decline only marginally (Jones et al., 1968; Kuttler and Adams, 1977; Corrier et al., 1981 ). Whether studies on post-infection immunity reflect protective mechanisms that may be induced by MSP immunization is unclear. Protection in outer membrane immunized cattle correlates with anti-MSP antibody titer (Tebele et al., 1991 ). However, this correlation does not imply a specific immune mechanism. Polyclonal antibodies, reactive with the MSPs, and monoclonal antibodies against the defined MSP- I a epitope have been shown to neutralize infectivity of isolated A. marginale for calves (Palmer and McGuire, 1984; Palmer et al., 1986a, 1987). However, these experiments used large quantities of antibody incubated with the organism in vitro prior to inoculation and may not reflect in vivo neutralization. In contrast to strictly antibody dependent mechanisms, antibody against MSP-1 has been shown to enhance phagocytosis ofA. marginaleby bovine macrophages (Cantor et al., 1993). A role for macrophage activation with enhanced phagocytosis and killing would be consistent with studies indicating the importance of the spleen and cell-mediated immune mechanisms. Despite these studies, it should be emphasized that the mechanisms by which MSPs induce protection are unknown.

6. Future directions

The ability of multivalent outer membrane immunization to induce protection similar to premunition clearly provides a basis for vaccine development based on antigenically defined MSPs. The expression of recombinant MSPs may allow development of a first-generation multivalent recombinant vaccine that mimics the native outer membrane immunogen. Based on the enhanced immunogenicity of Quil A micellar structures, used in the experiments with the native outer membranes, more rigorously defined but structurally similar matrices designated immune stimulating complexes (ISCOMs) have been developed for antigen presentation (Morein et al., 1984). ISCOMs are hydrophobically stabilized complexes

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of Quil A and lipid that incorporate multimers of antigen in a defined ratio with the Quil A. ISCOMs have now been made incorporating native and recombinant antigens from bacteria, viruses, and parasites and have demonstrated consistent enhancement of antibody titer to membrane proteins (Morein et al., 1987; Sundquist et al., 1988; Trudel et al., 1989). Specifically with bacterial outer membrane proteins, ISCOM incorporation of Neisseria gonorrhoeae membrane protein PI increased IgG titer approximately 250% relative to liposome incorporated PI following two immunizations (Kersten et al., 1988). Significantly, the N. gonorrhoeae PI protein anchors in the ISCOM in the same conformation as in the native outer membrane. This retention of native conformation and the presentation of multimeric complexes appears critical for the enhanced immunogenicity of ISCOMs, resulting in induction of neutralizing antibody and protection not achieved using inactivated whole organism vaccines (Osterhaus et al., 1985). ISCOMs are stable, lyophilizable, relatively easy to prepare and characterize, and non-toxic in cattle (Trudel et al., 1988). These characteristics, combined with the demonstration that the Quil A-A. marginale outer membrane immunogen induced protective immunity, support the use of ISCOMs for development of a first-generation vaccine. However, the long-term goal of protecting against multiple co-endemic hemoparasitic infections using a multivalent vaccine will require more careful selection ofA. marginale antigens. Although vaccine vectors with relatively large capacity for multiple antigen expression exist, the need to express antigens for each of several pathogens and, very possibly, cytokines for priming of important immune cell function will limit inclusion to those B and T cell epitopes absolutely required for protection. Certainly the ability of single recombinant MSPs to induce protection, although less effectively than the multi-MSP outer membrane immunogen, indicates that more defined subunits can induce relevant immune responses. Selection and optimization of recombinant based vaccines will require identification of the critical B cell and T cell epitopes on individual MSPs. In addition, our understanding of immune protective mechanisms must be substantially increased. Identification of the immune mechanisms at the molecular level will allow improved targeting of relevant T cell subsets and B cell responses to enhance the critical cellular and humoral mechanisms. 7. Babesia bigemina and Babesia bovis: Definition of vaccine antigens Two general areas in which our lack of understanding continue to limit the rational design of an effective antigenically defined vaccine against babesial parasites are the nature of the protective immune response and relevant antigen or epitope identification. These are not mutually exclusive areas, as a better understanding of protective immunity will certainly guide identification of key antigens. Likewise, logical or empirical identification of antigens which are capable of inducing partially protective immunity can aid in understanding the nature of immune protection on a defined epitope basis.

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Classical passive transfer experiments using either immunoglobulin or whole and fractionated spleen cell preparations indicate a role for both B- and T-lymphocytes in protective immunity against Babesia spp. (Mahoney, 1967b; Mahoney et al., 1979; Zivkovic et al., 1983/1984; Meeusen et al., 1984). Neither broad branch of the immune system alone seems capable of conferring complete homologous and heterologous strain protection. There have been no definitive experiments that clarify the relative role of CD4 and CD8 T-lymphocyte subsets in protective immunity, and neither the presence nor significance of a TH 1 or TH2 subset bias in immune animals has been investigated. However, T cell lines and clones generated from the peripheral blood of Babesia boris and Babesia bigemina immune cattle establish the presence of antigen specific TH0 and TH 1 lymphocytes (Tetzlaff et al., 1992; Brown et al., 1993a,b). Further, the ability of inflammatory cytokines and reactive nitrogen and oxygen intermediates to directly affect the viability of protozoan parasites indicates the potential importance of a TH 1 response in protective immunity (Clark et al., 1990; Green et al., 1990; Mendis et al., 1990; Taylor-Robinson et al., 1993 ). As this potential is verified and the contribution of other T-lymphocyte subsets is investigated, vaccine antigen selection will undoubtedly be influenced. The identification of epitopes important for specifically activating T-lymphocytes has recently been reviewed (Brown and Rice-Ficht, 1994). The constraints imposed on antigen selection by a multivalent approach also dictate a precise definition of B-cell epitopes relevant to induction of protective immunity. This level of understanding inevitably requires not only that antigens be identified, but also that amino acid sequences comprising B-cell epitopes be determined. With this goal in mind, the history and current status of different approaches toward synthetic vaccine development for Babesia spp. will be briefly reviewed. The use of sequence homologies to identify additional candidate vaccine antigens through an epitope directed approach will then be examined, using apical complex molecules as an example. 8. Antigen selection: Prior studies and current status

Several historical approaches for identification of candidate babesial vaccine antigens teach us valuable lessons about targeted antigen selection. These include the use of soluble exoantigens found in the supernatant of continuous cultures, selection of gene products binding to antibodies in immune sera, continuous refinement of protective crude antigen fractions to the individual polypeptide level, and identification of antigens by virtue of their potential function in invasion and accessibility to antibody. Continuous culture supernatant antigens can induce variable degrees of protective immunity against both Babesia boris and Babesia bigemina, indicating that a killed antigen preparation is capable of at least partial protection (Smith et al., 1981; Timms et al., 1983; Montenegro-James et al., 1987). While these results reinforce the potential of a defined antigen vaccine, the antigenic composition ofsupernatants is complex (sensitive techniques dem-

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onstrate many if not most of the same antigens present in killed merozoite preparations; T.F. McElwain, unpublished data, 1988); little light is shed on specific antigens responsible for inducing the partially protective immune response. Classical protein fractionation techniques coupled with monoclonal antibodies have been used to dissect complex mixtures of antigens that are capable of inducing partially protective immunity (Wright et al., 1992). This technique has resulted in the definition of three candidate vaccine antigens designated 12D3, 11C5, and 21B4, each capable of inducing a partially protective immune response against Babesia bovis (Wright et al., 1992 ). Similar immune protection against Babesia bigemina using homologues from this species has been reported, but detailed studies have not yet been published. While in the fractionation approach, the complex nature of the starting material obviates the possibility of testing each antigen individually, the data from these studies indicate that the relevant antigens cannot be selected solely based on their immunodominance (Wright et al., 1992). Several additional babesial antigens have been identified by virtue of their immunodominance during natural infection (Wright et al., 1983; Goodger et al., 1986; Gill et al., 1987; Timms and Barry, 1988). The inability of those immunodominant antigens that have been tested to induce protection (Wright et al., 1983; Goodger et al., 1986; Gill et al., 1987; Timms and Barry, 1988; Wright et al., 1992) further supports the argument that this criterion cannot be used alone to select vaccine candidates. However, general statements regarding the selection of whole gene products may be misleading. Two of the antigens selected by fractionation (21B4 and 11C5) have an immunodominant region that apparently diverts the immune response from critical regions in other parts of the molecule (Wright et al., 1992). In addition, an antigen associated with the apical complex (see below) and designated Bb-1 was initially identified by immunodominant reactivity with antibodies in immune serum (Trippet al., 1989). The dominant B-cell epitopes of Bb-1 reside in the C-terminal half of the molecule, which contains 28 tandem tetrapeptide repeats. However, sequences in the N-terminal half of the molecule comprise Thl epitopes that may be important to induction of protective immunity (Brown et al., 1993b). Thus, while immunodominant epitopes may not be effective as protective immunogens, antigens containing these epitopes may contain regions important for inducing protection. Candidate babesial vaccine antigens have also been selected based on their potential functional role and location. Antigens on the surface of the infected erythrocyte, merozoite, and sporozoite are accessible to antibodies and are likely involved in critical functions of nutrient acquisition, sequestration (in the case of Babesia boris), and host cell invasion. The vaccine potential of sporozoite surface antigens has not been tested. Several candidate molecules on the surface of Babesia boris and Babesia bigemina infected erythrocytes have been identified (Howard et al., 1980; Allred et al., 1993; Shompole et al., 1994), Interestingly, the 11C5 antigen identified in a protective antigen fraction of Babesia boris and later demonstrated to induce partial protection was initially selected in part be-

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cause of its association with the infected erythrocyte membrane (Goodger et al., 1992), lending credence to this approach for antigen selection. By analogy with malarial parasites and as found in studies with Babesia microti (Rudzinska et al., 1976 ), the process of invasion oferythrocytes by babesial merozoites involves several morphologically defined steps of initial association, tight attachment, and invagination with formation of the parasitophorous vacuole. Candidate Babesia bovis and Babesia bigemina vaccine antigens have been selected based on their putative involvement in this process since blocking of any step by an appropriate targeted immune response would prevent invasion. Merozoite antigens exposed on the surface are logical vaccine candidates, and a number of Babesia boris and Babesia bigemina merozoite surface antigens have been identified (McElwain et al., 1987; Goff et al., 1988; Hines et al., 1989; Reduker et al., 1989; Figueroa et al., 1990 ). Three of the Babesia bigemina surface proteins designated gp45, gp55, and p58 are capable of inducing an immune response that is partially protective, significantly reducing the peak parasitemia in experimentally challenged cattle (McElwain et al., 1991 ). Monoclonal antibodies against Babesia bigemina merozoite surface proteins and monospecific antibodies against the immunodominant merozoite surface protein of Babesia bovis (MSA-1) can significantly inhibit the invasion of merozoites in vitro (Figueroa and Buening, 1991; Hines et al., 1992). The inability of the same cattle from which the in vitro neutralizing Babesia bovis MSA- l monospecific antibodies were obtained to effectively resist experimental challenge (Hines et al., 1995 ) indicates that mechanisms ofmerozoite neutralization in vivo are different and probably more complex than what takes place in continuous culture. Nevertheless, taken together these experiments with both Babesia bigemina and Babesia bovis merozoite surface proteins suggest they have a critical role in some phase of merozoite entry and survival. In addition, while Babesia bovis MSA-1 is another example of an immunodominant antigen that is not capable of inducing protection in vivo, the ability of MSA-1 specific antibodies to neutralize infectivity in vitro reinforces the need to investigate immunity on an epitope rather than a whole antigen basis.

9. Antigen selection using an epitope directed approach If ligand receptor interaction of the merozoite and erythrocyte surface is important for invasion, conservation of critical domains within surface protein gene families would be reasonably predicted and should be reflected in sequence homologies. However, of the surface proteins investigated, few are both genetically and antigenically conserved on a broad basis. Marked polymorphism of the major merozoite surface proteins is the rule rather than the exception. There is a total lack of antigenic cross-reactivity among MSA-1 and MSA-2 gene products ofBabesia bovis, and among gp45 or gp55 surface proteins ofBabesia bigemina, from broadly disparate geographic strains (McElwain et al., 1991; Palmer et al., 1991; Jasmer et al., 1992; unpublished data). Yet rapid antigenic variation has

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not been demonstrated, and antibodies obtained during persistent Babesia bovis infection one year after challenge continue to react to immunodominant epitopes of MSA-1 from the original biologically cloned strain used to infect the cattle, indicating continuous stability of these epitopes (T.F. McElwain, unpublished data, 1994). Interestingly, even though sequence conservation of Babesia bovis MSA-1 among different strains occurs in the 3' end of the gene, a reading frame shift results in amino acid variation (Hines et al., 1992). Thus, if these gene products are indeed important in erythrocyte invasion, their role may be independent of precise sequence, but dependent on overall composition and charge, perhaps facilitating initial loose attachment. Earlier studies demonstrating the shedding of the surface coat of Babesia bovis (presumably consisting at least in part of MSA-1 and MSA-2) shortly after initial attachment (Rudzinska et al., 1976) support this hypothesis. However, even though their function may be critical to erythrocyte penetration, the marked antigenic polymorphism of MSA-1 and MSA-2 renders them less likely vaccine candidates unless specific conserved and critical epitopes can be identified. In contrast to most babesial merozoite surface proteins, a 58 kDa merozoite membrane molecule of Babesia bigemina and a similar 60 kDa molecule of Babesia boris are antigenically conserved among all geographic strains tested (McElwain et al., 1987; Palmer et al., 1991; Suarez et al., 1994a). The capability of both the 58 kDa molecule of Babesia bigemina and the 60 kDa molecule of Babesia bovis to induce partial immune protection against experimental challenge in cattle has been demonstrated (McElwain et al., 1991; Wright et al., 1992 ). Immunoelectron microscopy indicates that both these polypeptides are found in the rhoptries (Machado et al., 1993; Suarez et al., 1993), organelles that are one component of the apical complex, and they have thus been termed rhoptry-associated proteins 1 (RAP-1). The apical complex is a unique collection of organdies---including the rhoptries and micronemes in both Babesia bigemina and Babesia bovis, dense granules in Babesia bigemina, and possibly the spherical body in Babesia bovis--and is the defining feature of all apicomplexan parasites. The functions of this organellar complex are multiple and include a role in host cell invasion (reviewed by Perkins, 1992; Howard and Pasloske, 1993), but are poorly defined on a molecular level. It is clear in both Plasmodium and Babesia spp. that gene products found in the organelles are capable of inducing partial protection in immunization trials (McElwain et al., 1991; Perkins, 1992; Wright et al., 1992; Howard and Pasloske, 1993), and thus should be considered candidate antigens for vaccination. Again, it is reasonable to hypothesize that the remarkable conservation of apical complex organelles throughout this phylum translates into similar conservation of function, and consequently to conservation on a molecular level. Comparison of the nucleotide and amino acids sequences of Babesia bigemina and Babesia bovis RAP-1 indicates significant homology, particularly in the N-terminal 300 amino acids (Suarez et al., 1991 ). A similar level of homology can be found in genes from Babesia ovis and Babesia canis, defining a RAP-I gene family (Dalrymple et al., 1993 ). Sequence identity is limited to short oligopeptides

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with rather marked sequence variation in other parts of the molecules. Computer assisted analysis of more broad sequence conservation among apical complex polypeptides from erythroparasitic protozoa also indicates significant conservation (Suarez et al., 1991 ). More detailed comparison of Babesia bigemina and Babesia boris RAP-I sequences with the malarial AMA1/Pf83/Pk66 gene family identifies significant sequence similarity within small regions of the molecules, again owing to the conservation of short oligopeptides (Suarez et al., 1994b). Statistical analysis using computer derived oligopeptide consensus sequences based on all the RAP-1 and Pf83 gene family members indicates that several of these oligopeptides are specifically conserved and uniquely present. Such broad sequence conservation across genus lines logically suggests conservation of function, which for the surface-exposed babesial RAP-1 and malarial AMAI/Pf83/ Pk66 gene products may be a role in host cell invasion. The ability of these molecules to induce antibodies that are capable of blocking invasion in vitro (Figueroa and Buening, 1991; Thomas et al., 1984, 1990), and of inducing a partially protective immune response in vivo (Deans et al., 1988; McElwain et al., 1991; Howard and Pasloske, 1993 ), is further indication of the critical role they play in parasite survival. Despite overall sequence identity of 45% in the N-terminal 300 amino acids of Babesia bigemina and Babesia boris RAP-1, cross reactivity of B-cell epitopes can be demonstrated only with very high-titered monospecific serum antibodies (Suarez et al., 1993 ). No cross-reactivity is apparent with immune serum antibodies. Monoclonal antibodies against each RAP-1 gene product also do not cross react, and neither immune serum nor 12 newly characterized monoclonal antibodies prepared against isolated rhoptries ofBabesia bigemina (O. Vidotto et al., unpublished data, 1994 ) bind to Babesia boris. Thus, the sequence conservation recognized in RAP-1 gene products and presumably present in other rhoptry associated molecules does not translate into antigenic cross-reactivity. Precise mapping of RAP- 1 B-cell epitopes bound by monoclonal antibodies provides a basis for understanding this observation, as none of these monoclonal antibody defined epitopes corresponds exactly to a conserved motif (Suarez et al., 1993 ). In addition, peptide specific immune serum prepared against and strongly reactive with a strictly conserved RAP-1 14mer is unreactive with native RAP-1, suggesting that this isolated B-cell epitope is poorly immunogenic and inaccessible in the native molecule. Thus the conserved motifs do not appear to comprise linear Bcell epitopes in their native context. Their contribution to T-cell epitopes has not been investigated, nor has their precise role in the tertiary structure and function of the whole molecule been defined. While these studies once again illustrate the immunodominance lesson demonstrated in earlier experiments, they also provide an understanding of the molecular basis for this lesson, and reinforce the notion that identification of relevant vaccine components may be best accomplished on an epitope basis.

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10. Future directions Selection of candidate babesial vaccine antigens has been successfully accomplished with both classical fractionation techniques and the logic of location and putative function. Valuable lessons have been learned. However, neither approach has yet provided a basis for understanding the nature of the protective immune response or the selection of additional epitopes necessary for inducing complete protection against clinical disease. Paradoxically, the limitations of practicality and economics in lesser developed countries suggests that only the most scientifically refined multivalent vaccine against tick-borne diseases of livestock will be acceptable in endemic regions. Precise antigen definition will be critical. Understanding the relationship of conserved oligopeptides to tertiary structure, to function, and to B- and T-cell epitopes may provide a molecular basis for antigen selection with broad application. As progress is made in defining the host response that is important in immune protection, the convergence of these two lines of research should enable rational vaccine design from a solid scientific basis.

Acknowledgments The original research referenced by the authors was conducted in collaboration with Drs. William C. Davis, Stephen A. Hines, Douglas P. Jasmer, Donald P. Knowles, Rosangela Z. Machado, Travis C. McGuire, Lance E. Perryman, Fred R. Rurangirwa, Carps E. Suarez, Steven M. Thompson, OdiUon Vidotto, Marilda Vidotto, and Elizabeth Visser at Washington State University; Drs. Will Goff and David Stiller at the Agricultural Research Service, U.S. Department of Agriculture; Dr. Antony J. Musoke at the International Laboratory for Research on Animal Diseases; Dr. S.P. Shompole at the Kenya Agricultural Research Institute; Drs. Eugene Pipano and Varda Shkap at Kimron Veterinary Institute; Dr. Katherine M. Kocan at Oklahoma State University; Drs. Wendy Brown and Allison Rice-Ficht at Texas A&M University; Drs. A.R. Alleman, David R. Allred, Anthony F. Barbet, Gamal E. Eid, Vishnu S. Mishra, Suzan M. Oberle, and Edward B. Stephens at the University of Florida; Dr. Gerald Buening at the University of Missouri; and Drs. Ntando Tebele, Devere Munodzana and Tendai Ushe at the Zimbabwe Veterinary Research Laboratory. We appreciate the access to unpublished research and manuscripts submitted or in press provided by Drs. Alleman, Allred, Barbet, Brown, Knowles and McGuire. The original research by the authors has been supported, in part, by the United States-Israel Binational Agricultural Research and Development Fund Project US-1855-90RC and US2238-92C; USDA NRICGP 92-37204-8180; US Agency for International Development grants DAN-4178-A-00-7056-00 and DHR-5600-G-00-1035-00, and the Washington Technology Centers.

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