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Veterinary Parasitology 101 (2001) 291–310

Advances and prospects for subunit vaccines against protozoa of veterinary importance
Mark C. Jenkins∗
Immunology and Disease Resistance Laboratory, Agricultural Research Service, US Department of Agriculture (USDA), Beltsville, MA 20705, USA

Abstract Protozoa are responsible for considerable morbidity and mortality in domestic and companion animals. Preventing infection may involve deliberate exposure to virulent or attenuated parasites so that immunity to natural infection is established early in life. This is the basis for vaccines against theilerosis and avian coccidiosis. Vaccination may not be effective or practical with diseases, such as cryptosporidiosis, that primarily afflict the immune-compromised or individuals with an incompletely developed immune system. Strategies for combating these diseases often rely on passive immunotherapy using serum or colostrums containing antibodies to parasite surface proteins. Subunit vaccines offer an attractive alternative to virulent or attenuated parasites for several reasons. These include the use of bacteria or lower eukaryotes to produce recombinant proteins in batch culture, the relative stability of recombinant proteins compared to live parasites, and the flexibility to incorporate only those antigens that elicit “protective” immune responses. Although subunit vaccines offer many theoretical advantages, our lack of understanding of immune mechanisms to primary and secondary infection and the capacity of many protozoa to evade host immunity remain obstacles to developing effective vaccines. This review examines the progress made on developing recombinant proteins of Eimeria, Giardia, Cryptosporidium, Toxoplasma, Neospora, Trypanosoma, Babesia, and Theileria and attempts to use these antigens for vaccinating animals against the associated diseases. Published by Elsevier Science B.V.
Keywords: Vaccine; Recombinant antigen; Eimeria; Giardia; Cryptosporidium; Toxoplasma; Neospora; Trypanosoma; Babesia; Theileria

1. Introduction Veterinary parasitic diseases cause enormous annual economic losses to livestock industries worldwide (for review, see Willetts, 1994). Although many of these diseases are
Tel.: +1-301-504-8054; fax: +1-301-504-5306. E-mail address: mjenkins@anri.barc.usda.gov (M.C. Jenkins). 0304-4017/01/$ – see front matter. Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 5 7 - X

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M.C. Jenkins / Veterinary Parasitology 101 (2001) 291–310

drug-treatable, the capacity of protozoan parasites to rapidly develop drug-resistance has prompted the search for alternative means of prevention. The cost of drug treatment can also be prohibitive, especially in developing countries where many of the diseases are commonly found. The nearly complete immunity that is elicited during a primary infection with many protozoa has been the basis for vaccine development. In fact, inoculation of susceptible animals with either virulent or attenuated parasites has been a successful approach to vaccination against several protozoan diseases. There are, however, several drawbacks to using live parasites, not least of which are the need for cold storage, the limited shelf-life of the vaccine, the possibility of causing morbidity and mortality in vaccinates, and the risk of attenuated organisms reverting to a more pathogenic state. Subunit vaccines, derived from native antigens of the parasite or as recombinant proteins from cloned DNA, may overcome these difficulties. Rapid advances in our understanding of mechanisms of immunity to many protozoa and the development of molecular tools for generating vaccines comprised of recombinant antigens or cloned DNA itself bodes well for vaccine development in the near future. This expectation must, nonetheless, be tempered by the reality that many protozoa have developed elaborate mechanisms (e.g. antigenic variation) for evading host immunity and that primary infection with one strain may not protect against heterologous strains. The display of highly immunogenic surface antigens that provoke “non-protective” responses or the secretion of products that cause immunopathology are other examples of protozoan adaptation to host immunity. Thus, the task of researchers seeking to develop vaccines against protozoa is to identify antigens that elicit responses which will destroy the parasite rather than exacerbate the deleterious effects of infection. The difficulty is that, generating a protective immune response is dependent on a number of complex interrelated factors. Variables such as antigen composition, processing, and delivery, the need for adjuvant and booster immunization to promote a particular immune response, and the age of host animal all impact the efficacy of vaccination. Also, as the antigenic complexity of a recombinant vaccine increases, so does the cost of production such that it may no longer be competitive with drug treatment. As insight is gained on immunity to protozoa and on the developmental stages and associated antigens targeted by the protective response, then molecular and immunological tools may be harnessed for generating effective vaccines. What follows is a review of efforts to develop subunit vaccines based on recombinant antigens derived from cloned DNA of protozoan parasites of veterinary importance. Also included is a brief description of the immune mechanisms that are thought to play a role in controlling primary and secondary infections because eliciting similar responses with a recombinant antigen may be critical to vaccine efficacy. For purposes of this review, the protozoa are grouped into three categories related primarily to the parasite’s life cycle. 2. Homoxenous, non-cyst-forming protozoa: Eimeria, Giardia, and Cryptosporidium 2.1. Eimeria Coccidiosis in avian and mammalian hosts is primarily an intestinal disorder characterized by poor weight gain and inefficient feed utilization due to alteration of the intestinal mucosa by protozoa in the genus Eimeria. Although immunovariability has been observed among strains of E. maxima, infection of poultry with Eimeria spp. oocysts usually re-

. 1991. but is probably related to either the use of a “non-protective” antigen or to inappropriate delivery of the vaccine to the mucosal immune system. or drug-arrested Eimeria spp. Delivery of recombinant Eimeria antigens using Salmonella. 1993a. 1995. IFN. 1976. 2000). Williams. 1998. falciformis.C. Jenkins et al.b). Recombinant antigens of avian coccidia have generally been derived from cDNA originating from mRNA of extracellular stages of the parasite (sporozoites. 1998. vermiformis.b.. Stiff and Vasilakos. Vermeulen. papillata have shown that CD4+ T cells and associated cytokines. and E.b. 2. Research in mouse models using E. 1999). These vectors have been shown to elicit CD4+ and CD8+ T cell responses in infected cells (Wang et al. whereas CD8+ T cells are involved in immunity to secondary challenge (Rose et al. fowlpox virus (FPV). Giardia intestinalis) by prior exposure to the parasite . Jeurissen et al. It is likely that CD4+ and CD8+T cell responses during primary and secondary infections are directed against antigens expressed by parasite-infected cells. gametes) of Eimeria spp. Rose et al. Supporting this idea is a recent finding that recombinant E. 1998). There is also evidence for merogonic stages inducing and being targeted by protective immunity (Rose and Hesketh. Lillehoj and Lillehoj. Williams et al. Jenkins / Veterinary Parasitology 101 (2001) 291–310 293 sults in complete. the latter attenuated by the absence of several merogonic stages (Shirley et al. merozoites. 1976.. irradiated.. 1988). These DNA sequences have been expressed in a variety of prokaryotic and eukaryotic vectors. 1990. 1998). Ramsay et al. The reason for failure to stimulate protective immunity is unknown.. 1973. Current vaccines consist of a mixture of virulent or precocious strains of coccidia. Recent studies in chickens indicate that immunity to primary and secondary Eimeria infections is similar to that observed in mice (Lillehoj and Trout. resistance against challenge (Joyner and Norton...and IL-12 are important for controlling primary infection.M.. tenella sporozoite antigen was protective only if administered to chickens by direct DNA vaccination (Kopko et al. merozoites). 1996. or herpesvirus of turkeys (HVT) expressing Eimeria DNA sequences may be a viable approach to protect chickens against coccidiosis (Vermeulen. 1998. Protective immunity against coccidiosis will probably require the processing and presentation of recombinant antigen in conjunction with MHC molecules on the surface of host cells for inducing appropriate effector mechanisms that prime the host against subsequent parasite challenge. see Jenkins. 1997). 1989a. 1985.2. suggests that sporozoite-infected cells are necessary and sufficient for the development of protective immunity (Jeffers and Long. 1996.. 1986. E. 1999). Cronenberg et al. but immunization has only elicited partial protection against oocyst challenge (Jenkins. Studies of resistance elicited by infection with virulent.. (for review. 1991a. Stiff and Bafundo. 1993). the immunizing inoculum must be generated in chickens followed by collection and processing of fecal material for oocysts. Schito and Barta. 1998. 1999). Thus.. Vermeulen. intracellular processing would be required for the presentation of recombinant antigen to the avian immune system. 1998. Giardia There is ample evidence that humans and animals develop immunity to Giardia duodenalis (synonyms: Giardia lamblia. While these vaccines are effective in protecting broilers against coccidiosis. McDonald et al. 2000). Several laboratories over the last two decades have attempted to develop a subunit coccidiosis vaccine using recombinant antigens expressed from DNA of various developmental stages (sporozoites.. Long et al. albeit species-specific.

based on studies in humans and mice. Jenkins / Veterinary Parasitology 101 (2001) 291–310 (reviewed in Heyworth. 1985. Studies in mice indicate that CD4+ T cells play an accessory role in B cell secretion of IgA and protection against G. DNA encoding a 32 kDa flagellar protein (Upcroft et al. whereas chronic disease. IFN. or antigens associated with the ventral disc. vaccination of the most vulnerable individuals (the young) would probably not be effective during the period of peak susceptibility. 1997). 1988). duodenalis-cell culture (Olson et al. duodenalis sequences against giardiosis has not been tested.. IgA. marked by unremitting diarrhea and shedding of Giardia cysts. 1995. Several studies have shown that passive immunotherapy with hyperimmune serum or colostrum against C. but attach to the host intestinal epithelium. see Riggs.. Cryptosporidium Cryptosporidiosis causes a self-limiting diarrhea in humans and animals that resolves within 1–2 weeks post-infection (O’Donoghue. 1992.and IL-12 (for review. see Riggs. 1999). Prior exposure to Cryptosporidium parvum leads to increased. the role of these antibodies in resolution of cryptosporidiosis remains unclear (for review.. the cloning and expression of antigen genes for vaccine testing has only recently begun. Protective immunity appears. 1992). 1998). 2. Fayer et al. but not complete resistance. duodenalis infection by preventing the attachment of trophozoites to host cells (Kaplan and Altmanshofer.. to involve CD4+ T lymphocytes and associated the cytokines. due to the sporadic occurrence of cryptosporidiosis... 1992) have been described. Faubert. Second. MHC-restricted CD4+ and CD8+ T lymphocyte responses do not appear to be a primary effector mechanism in immunity. Heyworth. 1997).. parvum-specific IgG.. and since . Although C. is commonly observed in the young or persons with hypogammaglobulinemia (Heyworth. Deguchi et al.. 1989).. It is believed that humoral components. Olson et al. in particular secretory IgA. 1998). However.. 2000). see deGraaf et al. muris (Heyworth et al.294 M. 1995). There are several reasons for this approach. the protective efficacy of recombinant VSP antigen or other expressed G.. 1992. The disease is prevalent in the young and in immunocompromised persons due to immature or impaired T cell immunity (Casemore et al. VSP was recently expressed in a vaccine strain of Salmonella typhimurium and used to elicit VSP-specific serum IgG and intestinal IgA responses in mice (Stager et al. duodenalis trophozoite extracts (Vinayak et al. 1987.3. parvum antigens can ameliorate clinical signs of disease (reviewed in Jenkins et al. 1987).C. kinetosomes and funis. 1987) or a variable surface protein (VSP) (Aggarwal et al. Vaccination against cryptosporidiosis has focused on antigens that may be used to generate colostrum for passive immunotherapy rather than stimulating protective immunity (for review. are involved in resolution of G. and IgM responses have been demonstrated. 1997). possibly because trophozoites do not actually invade cells. 1987). or anterolateral axonemes have also been reported (Upcroft et al. Inge et al. the age-related resistance to infection. Although animals can be vaccinated against giardiosis by immunization with G. 1999).. duodenalis-specific IgG can directly lyse the parasite by both complement-dependent and -independent means (Belosevic and Faubert. 1996). 2000) or supernatant from G. Other less well-characterized clones encoding a surface coat protein. First. to subsequent infection (Chappell et al. 1989.. There is also in vitro evidence that G. Nash and Mowatt.. 1987. Giardiosis is a self-limiting infection in immunocompetent persons.

. Alexander et al. Priest et al.. Cevallos et al. 1997. Tosini et al. gondii results in complete resistance to secondary challenge (Araujo. 1998) against recombinant and native CP15 antigens. parvum DNA has concentrated on expressing epitopes on the surface of invasive stages (sporozoites. This resistance is due in part to the presence of bradyzoites/cysts in many tissues. 1997. Another study showed that administration of monoclonal antibodies (mAbs) specific for a 23 kDa C. 1996)... a recent study showed that expression of CP15/60 in an eukaryotic vector (baculovirus) was superior to a prokaryotic system (E. Although it is unknown whether direct plasmid DNA injection is required. gondii infection (Uggla and Buxton. 1999a).C. Thus. there has been considerable interest in developing vaccines against productive T.. Jenkins / Veterinary Parasitology 101 (2001) 291–310 295 disease symptoms are rarely life-threatening.M.b. 1993a.. Spano et al. immunization of ruminants with either recombinant C... fatal CNS disease in immunosuppressed persons) as well as causing abortion in sheep and goats. there is little perceived need for widespread vaccination. 1996). in the latter host species..1. 1999). Heteroxenous. Thus. 1998. Plasmid DNA expressing the CP15/60 protein has been used to elicit serum and colostrum antibodies in sheep (Jenkins et al.. 1999). parvum sporozoite surface antigens or plasmid DNA encoding the CP15 or CP23 antigens appears to be a viable approach to producing colostrum for the passive immunotherapy of cryptosporidiosis. CP15-specific T cell proliferative responses were also demonstrated in both the spleen and the mesenteric lymph nodes (Sagodira et al. Vaccine development is based on the observation that primary exposure to T. 3. tissue cyst-forming coccidia: Toxoplasma. remained high for at least 1 year post-immunization (Sagodira et al. parvum antigens have been described (Lally et al. 1994. 1999.. 2000). cloning of C. 1990. coli) in terms of humoral and cellular responses elicited (Iochmann et al... In mice. Neospora 3. 1995) and cows (Jenkins et al. merozoites) which may be involved in attachment to host cells of the intestinal epithelium. parvum sporozoite protein reduced the severity of C. 1999a)..g. Khramtsov et al. Treatment of immunosuppressed adult inbred mice with this anti-CP15/60 hyperimmune bovine colostrum (HBC) prior to and during experimental C.. Dubey. Perryman et al. 1999b). 1996. kids challenged with a high number of C.. congenital toxoplasmosis. parvum oocyst infection in mice (Perryman et al. 2000). 1996). Nasal immunization of goats and mice with a second CP15 DNA sequence also led to anti-CP15 antibodies in serum that.. 1998). parvum oocysts (Perryman et al. the greatest effort has been directed at sequences coding for antigens related to sporozoite 15 or 23 kDa surface proteins (Jenkins et al. Although a number of recombinant C.. HBC prepared against recombinant CP23 antigen prevented diarrhea and reduced oocyst shedding in calves challenged with 106 C. Furthermore. parvum oocysts (106 oocysts per kid) and fed colostrum from CP15-immunized goats shed fewer oocysts and displayed less severe clinical effects compared to kids fed normal colostrum (Sagodira et al. 1992. A role for tachyzoites in immunity is indicated by the success of commercial vaccines . Barnes et al. particularly in muscle and nerve cells.. parvum oocyst infection hindered parasite development in vivo (Jenkins et al. Toxoplasma gondii Due to the impact of toxoplasmosis on human health (e.

1999. but not against cyst challenge (Lunden et al.. Duguesne et al. Supply et al. 1998). Petersen et al.. While the innate immune response to T. mice immunized with pSAG1 showed higher survival rates and contained fewer brain cysts after cyst challenge (Angus et al. 7) or rhoptry (ROP4) antigens (Nielsen et al.. 4. Toxovax) that are derived from non-cyst-forming mutant strains of T. 1996. Lunden et al.g. Several studies have shown that mice and rats immunized with whole T. Darcy et al. 1996). which conflicts with another study that found 80–100% survival of pSAG1-immunized mice (Nielsen et al. the adaptive immune response appears to depend on CD8+ T cells activated by IL-2 secretion from CD4+ Th1 cells that have been activated by costimulatory molecules IFN. pSAG1 mice were not protected against tachyzoite challenge. Higher rates of survival and fewer brain cysts were observed in mice immunized with .. 1999. Angus et al. Vercammen et al.296 M... 2000. 1998). 1999)... gondii is mediated by IFN. 1993. 1990.. Mice inoculated with SAG1 DNA-transfected lymphoma cells also showed good protection as measured by survival against tachyzoite challenge (Aosai et al. Araujo.. oocyst shedding is prevented in cats inoculated per os with mutant T263 cysts/bradyzoites (Frenkel et al. 1998. 1993.. protective effects were enhanced by direct immunization with plasmid DNA containing sequences for tachyzoite surface (SAG1). In one study.. 1993). 1999. gondii or different measures of protection. the data indicate that rSAG1 expressed in either E. Immunization with p30 (SAG1) and p22 (SAG2) tachyzoite antigens incorporated into ISCOMs protects mice against tachyzoite or oocyst challenge.5).and IL-12 (reviewed by Alexander et al. For instance. 1984. Nielsen et al. Aosai et al.. Based on these observations. or excretory-secretory antigens (ESA) conferred protection against tissue cyst or tachyzoite challenge as assessed by reduction in a number of brain cysts (Araujo and Remington. In one study. 2000).. Velge-Roussel et al.. 1999). Jenkins / Veterinary Parasitology 101 (2001) 291–310 (e.. 1996. but not by parenteral immunization with any stage of the same parasite (Freyre et al. 1996). In the same study. 2000. gondii tachyzoite extracts or specific native antigens such as SAG1 (p30). is that immunization with one developmental stage (or antigens associated with this developmental stage) does not always confer protection against other stages (Alexander et al. gondii life cycle will require vaccinating different hosts with antigens associated with the stages they are likely to encounter.and TNF -activated macrophages. coli-derived rSAG2 were unsuccessful (Lunden et al. Zenner et al. 1991). Debard et al.. GRA2 (gp28... researchers have utilized either recombinant tachyzoite antigens or plasmid DNA encoding tachyzoite antigens to immunize mice or rats against T. coli or BCG confers partial protection against tissue cyst or tachyzoite challenge (Letscher-Bru et al. 1993).. GRA5 (p21)... Relevant to the successful generation of attenuated or subunit vaccines against toxoplasmosis. gondii (Buxton.. 2000). Angus et al. gondii oocyst or tissue cyst challenge by immunization with E.. Although it is difficult to make comparisons between studies that utilized different strains of inbred mice or different strains and challenge inocula of T. mice immunized with rSAG1 in conjunction with alum showed higher survival rate against tachyzoite challenge (Petersen et al. 1999.. Immunization of mice with a mixture of rSAG1 and IL-12 appeared to elicit excellent protection against brain cyst formation relative to control animals or those immunized with rSAG1 alone (Letscher-Bru et al. The discrepancy in outcome between the two studies may be due to differences in the mouse strains and plasmid constructs used. 2000). 1998. These findings imply that breaking the T.C. Brinkmann et al. 1993).. 1999. Desolme et al. 1992. In general. 2000).. gondii. Attempts to protect mice against T. dense granule (GRA1.. 1994).

C. 1999). SAG1 (Nc-p29. Studies of natural and experimental Neospora caninum infections indicate that abortions are caused by tachyzoites that arise either from reactivation of bradyzoites/tissue cysts or from oocysts that have been ingested during pregnancy. NCDG1) (Lally et al. 2000). depending on gestational age. Augustine et al.and IL-12 appear to be involved in immunity to acute N. pGRA7-. 1999). 2000). GRA7. and GRA7 (p33. 1998). 1997. will require delivery of several antigens from T. 1998) have been described as well as clones for surface antigens SRS2 (Nc-p43. Complete protection against vertical transfer of N. the co-injection of mice with pGRA4 and pGMCSF enhanced protective effects. 1996. Bjorkman et al.2. DNA clones of dense granule proteins. In the latter study. Identifying which antigens are targeted by protective immunity and which are involved in parasite survival within the host cell is the goal of several laboratories. caninum infection in mice (Kahn et al... caninum tachyzoites concomitant with neutralizing mAbs to IL-4 transmitted negligible numbers of tachyzoites to their pups after virulent N.M. GRA2 (p29) (Ellis et al.. and ROP2 (Vercammen et al. Conflicting results have been found in cattle immunized with whole tachyzoite antigen. Nishikawa et al. GRA6 (p37.. Nc-p35) (Hemphill et al. GRA6. 1998). caninum infections of sheep (Innes et al. 1998). Neospora Although neosporosis has been described as causing neurological signs in canines.. Jenkins / Veterinary Parasitology 101 (2001) 291–310 297 plasmid DNA containing sequences for GRA1. The prospect for vaccinating cattle against neosporosis appears good. or GRA7 on the invasion of host cells in vitro by N.. gondii life cycle stages using vectors that mimic processing by antigen-presenting cells in primary and secondary infections. 1999). 2000) or GRA4 (Desolme et al. Although pGRA1-... horses. there appears to be a genetic effect on responses to T.. 2000).. 2000). Sonda et al. no protection was observed in either BALB/c or C57/Bl6 mice (Vercammen et al. 1998). Similar to findings in DNA vaccination against malaria (Doolan et al. which. 1991. and other animals. SRS2. gondii plasmid DNA injection. 1997. caninum tachyzoites was not prevented (Andrianarivo et al. 1997.. caninum tachyzoites challenge during pregnancy (Long and Baszler. 1996. Paré et al. Further evidence for Th1 cytokines. caninum tachyzoites (Hemphill. Baszler et al. Hemphill et al... 1998. Rapidly multiplying tachyzoites cross the placenta and infect the fetus... 2000a). 1995) and cattle (Lunden et al.. 3. 1999).. IFN.. such as cats or pigs.. 2000). NCDG2) (Liddell et al. Nc-p36) (Howe et al... congenital transfer of N. 1999. 2000) by this immunization regimen... 1996. Th1 responses and associated cytokines IFN.. Howe et al. 1998). These data indicate that development of a subunit vaccine against toxoplasmosis for protecting outbred animals.. . the primary impact of this protozoan disease is on the reproductive health of cattle (Dubey. caninum tachyzoites was achieved in mice that had been immunized with whole tachyzoite antigen prior to mating and parasite challenge (Liddell et al. 1997. may lead to abortion (Anderson et al. and pROP2-immunized C3H mice show enhanced survival and fewer brain cysts after tissue cyst challenge. A role for these dense granule and surface proteins in host cell invasion is the inhibitory effect of antibodies against SAG1. Although humoral and cellular immune responses were elicited (Andrianarivo et al. Williams et al. is a recent study which showed that mice injected prior to pregnancy with avirulent N.also appears to be important in N.

Immunity to T. cruzi challenge infection (Miller et al. is transmitted to both humans and animals by triatomin bugs. 1988. In recent studies. 1992. the causative agent of Chagas disease (American trypanosomiasis) in Central and South America. Theileria 4. brucei brucei. immunization of female mice with recombinant VV-SRS2 prior to mating conferred significant protection against congenital N... Antibodies binding to the surface antigens of T. 1976.. Wrightsman and Manning.. cruzi trypomastigotes and mixed with either Freund’s adjuvant. Mice injected with recombinant TS plasmid DNA mounted a Th1-type response.. cruzi. In vitro studies using sera and cells from pTSA-1 immunized mice showed specific responses to T. Costa et al. 1995. 1999). The first three are transmitted by the bite of tsetse flies and are the causative agents of a disease complex in ruminants known in Sub-Saharan Africa as nagana. T. DNA encoding other “protective” antigens of T. haemoprotozoa: Trypanosoma. in particular... genes for a 40 kDa microneme antigen (Sonda et al. cruzi may be identified by novel techniques such as immunization with whole genomic or cDNA libraries prepared in eukaryotic plasmid vectors (Alberti et al. but eventually succumbed to T. cruzi challenge. 2000). Wizel et al. cruzi. Recent studies showed that mice immunized with recombinant vaccinia virus (VV) or dogs immunized with canine Herpes virus expressing SRS2 elicited N. cruzi challenge (Pereira-Chioccola et al. evansi. and T. cruzi challenge by injection of plasmid DNA expressing the sequence for trypomastigote surface antigen 1 (TSA-1) (Wizel et al. congolense. have been used to elicit cellular immune responses in mice and 100% survival against T. 2000b. T. coli. and a subtilisin-like serine protease (Nc-p65) (Louie and Conrad. date-mated. . cruzi appears to involve the cytokines IFN. 2001). In addition. Trypanosoma Trypanosomes of veterinary importance include T. Dos Reis.1.and TNF secreted by CD4+ Th1 and CD8+ Tc1 lymphocytes (Silva et al. 1998. 2000).. 1997. to a membrane-bound transialidase (TS) appear to play a role in protective immunity by possibly blocking parasite invasion of host cells (Krettli and Brenner. or adenovirus expressing IL-12. Pereira-Chioccola et al. and then challenged with N.c). a TRAP homologue (NcMIC2) (Lovett et al.. cruzi. However. Babesia. vivax. Umekita et al. Paraflagellar rod (PFR) proteins derived from the flagellum of T. caninum tachyzoites resulted in only partial protection against congenital transfer of the parasite to offspring (unpublished observations).. Franchin et al. 1997).C. mice immunized with recombinant TS protein showed a typical Th2-type response and 60% survival after T. cruzi trypomastigotes and. 1999) have been reported. Preliminary trials in our laboratory wherein adult BALB/c mice were immunized with either Nc-GRA6 (rNCDG2) or Nc-GRA7 (rNCDG1) expressed in E. T. Studies are underway to test direct injection of plasmid DNA expressing Nc-GRA6 and Nc-GRA7 for eliciting higher levels of protection against congenital neosporosis. recombinant IL-12. 1998). 1998). 1997. 1998). T... Jenkins / Veterinary Parasitology 101 (2001) 291–310 In addition. mice that were immunized with either recombinant TS protein or plasmid DNA expressing TS showed reduced parasitemia in blood compared to control mice (Costa et al. caninum-specific immune responses (Nishikawa et al. 1996. 1999).298 M. Tarleton.. caninum infection (Nishikawa et al.. 2000). 4. Heteroxenous.. Others have elicited protective immunity against T.

T. immunization of cattle with native Babesia antigen extracts or culture-derived supernatants containing secreted Babesia antigens elicit protective immunity against both homologous and heterologous challenge (Smith et al. Identifying antigens that elicit complement-fixing antibodies. 1987. A major obstacle in identifying candidate vaccine antigens is that the surface of T.2. 1998. This difference may account for the higher levels of T. B. such as IgG1 . While T.. cruzi. Immunity to acute infection appears to involve macrophages ... 1998). It has been estimated that the T. congolense. 1987. Schleifer and Mansfield. 1999). congolense-specific IgG1 in resistant cattle (Taylor et al. but not in trypanosensitive cattle (Mertens et al. 1994). congolense remains confined to the vasculature. Taylor. The search for vaccine targets on the surface of trypomastigotes should. In the past. Babesia Although Babesia species have been described in dogs (B. congolense infections (Sileghem et al. the principal causative agent of babesiosis in US (also termed Texas red water fever). 1989). but also depressing cellular immune responses (Taylor and Mertens. therefore. Montenegro-James et al. bigemina. the vast majority of research has been conducted on B.. First. is increased in trypanotolerant cattle. Timms. cattle that recover from a primary Babesia infection or that have been immunized with attenuated parasites are resistant to challenge infection (Mahoney et al. Trypanotolerant cattle control both anemia and parasitemia better than trypanosensitive breeds of cattle. 1995). lymph nodes. Mabbott et al. and B. 1998. This immune suppression may be due to counter regulation by the Th2 cytokine. which causes bovine piroplasmosis in other parts of the world. 1999).. IL-10 mRNA is elevated in leukocytes in peripheral blood. bigemina and B. brucei and T. 1989. 1973. babesiosis has been controlled by application of acaricides or by infection of cattle with an attenuated Babesia vaccine. 1998). be focused on invariant molecules that are essential for parasite survival (Borst and Rudenko.. IL-10. Beschin et al. are transmitted to cattle by the bite of Boophilus ticks.. The repeated acaricide resistance in ticks and the costs associated with its use has limited acaricides as a control measure (Palmer and McElwain. Timms et al. Progress on developing vaccines against African trypanosomes has been hindered by the reality that these parasites have developed mechanisms for not only evading immunity through antigenic variation.. canis) and horses (B. Second. should be a goal of vaccine development against T. 1996). termed variable surface glycoprotein (VSG). Timms. the African trypanosomes are strictly extracellular parasites. 1983.. brucei genome contains as many as 1000 different VSG gene sequences and that VSG expression can be turned on or off coincident with the production of antibodies by the host against a specific antigenic type (termed variable antigenic type or VAT). 1999). 1979. IL-4. 4. and spleen (Taylor. 1981. 1994). the other species are able to invade tissues. Several findings support the development of a vaccine against babesiosis.C. congolense-infected cattle showed that mRNA for a second Th2 cytokine. bovis of cattle. brucei and T. bovis. Studies in T. As indicated by studies in cattle. equi). An overt depression of T cell proliferation and macrophage/monocyte responsiveness has been found in experimental T. brucei evades immunity by using at least three different mechanisms for switching VSG expression (Borst and Rudenko. Brown and Palmer. brucei trypomastigotes is covered with a highly immunogenic protein. 1993. Jenkins / Veterinary Parasitology 101 (2001) 291–310 299 In contrast to T.M.

In spite of belonging to a multi-gene family. bovis challenge (Wright et al. 1995. Hines et al. bovis (Wright et al. immunodominant native and recombinant B. see McKeever and Morrison. Protective immunity has also been achieved with native B. A similar series of events occur in T.300 M.. the developmental stage infectious for ticks (for review. 1992). there is sufficient epitope conservation between various B. 4... bigemina (Dalrymple. Jenkins / Veterinary Parasitology 101 (2001) 291–310 and NK cells. Mr45.. bigemina and B. also appears to confer significant protection against B. appears to cross-react between different strains of B. is the agent of tropical theileriosis which is found primarily in North Africa.. Although RAP-1 gene sequences are polymorphic. Jasmer et al. 1993). exhibits high levels of cross-reactivity and confers protection against babesiosis in cattle (Dalrymple et al. 1992.. and >1500 kDa have shown no capacity for eliciting protective immunity against B. 2000). that may have protease activity. other immunodominant proteins.. 1999). 1998. annulata. At the injection site. 200. bovis antigens of 70. elicited 90% protection against B. 1992). 1991. the causative agent of East Coast Fever (ECF) is found in Eastern and Central Africa and is transmitted to cattle through the bite of the three-host tick Rhipicephalus appendiculatus. after lysing the host cell. 1991). southern Europe. which is a spherical body protein (SBP-1) (Goodger et al. bovis RAP-1. parva...C. annulata is ixodid ticks of the genus Hyalomma. Wright et al...3. 1993) and confers protection against babesiosis. Boulter and Hall. 1992). annulata sporozoites enter monocytes. termed variable major surface antigens (VMSA). which is associated with rhoptries. Dalrymple et al. Mr55. . an Mr58-60 antigen. of merozoites have shown no protective efficacy in vaccine trials (reviewed in Dalrymple. A high Mr antigen (70–200 kDa) and a 38 kDa cysteine-rich protein. It is generally thought that an effective babesiosis vaccine should activate CD4+ T cells to secrete IFN.. Brown et al.. 1992. IL-12. Dalrymple et al. and Central Asia including India. Theileria Piroplasms in the genus Theileria cause significant impact on cattle production in developing countries throughout the world (Uilenberg et al. parva. Brown and Palmer. B. 1993). 1992). bigemina merozoite antigens Mr72. A second important species. named Mr225 (BvVA1). Likewise. T.. The principal vector of T. Brown and Palmer. T. 1993). undergo schizogonous replication to generate large number of merozoites which. 1992. Conversely. 1995) and with native and recombinant Mr60 rhoptry protein (RAP-1. and TNF . 1993. the Middle East. 1993. A third recombinant protein. It is possible that VMSAs such as MSA-1 (42 kDa) and MSA-2 (44 kDa) are ineffective because they are encoded by a polymorphic gene family. and Mr36 have shown protective efficacy in cattle (McElwain et al. bovis Mr77-80 (Bv80). A number of different approaches have been taken to identify protective antigens associated with merozoites or merozoite-infected erythrocytes of B. 1999). T.that in turn elicits a neutralizing and complement-fixing IgG response and activates macrophages to produce the inflammatory cytokines IL-1. while the immune response that controls persistent infection involves activated macrophages and cytophilic Ab (Palmer and McElwain. bovis strains to suggest that this recombinant protein is a good candidate for inclusion in a babesiosis vaccine (Suarez et al. Bv60) (Wright et al. bovis. which appear to be neither abundant nor immundominant.. bovis challenge (Timms et al. which is a homologue of B. 1988. bovis merozoite antigen. Another recombinant B. invade erythrocytes and become piroplasms.

Immunization of cattle with SPAG-1 expressed as a fusion protein with a hepatitis virus B core antigen elicited antigen-specific antibodies and CD4+ responses as well as partial protection against T. annulata are not thought to play a major role in resistance. not least of which is the expense of vaccination and the requirement for cold storage to preserve vaccine integrity. The pathology of T. parva sporozoite antigen (p32) and a merozoite/piroplasm antigen (mMPSA) failed to elicit a protective response (Skilton et al. 1999). 1998). 1992). parva and the SPAG-1 (sporozoite antigen 1) of T.C. 1997. The use of VV-p67 alone or VV-p67 with VV expressing IL-4 did not provide immunity against T. high Ab titers that neutralize sporozoites in vitro occur after primary infection (Musoke et al.. annulata have similar life cycles. For instance. parva (Musoke et al.. Preston et al. TAMS1-1 and TAMS1-2.. 1994.. coli as a fusion protein to influenza virus NS1 showed partial protection against sporozoite challenge (Musoke et al. annulata sporozoite challenge (Boulter et al. Although both T. Baldwin et al... McKeever et al.. 1988). Similar to acaricide tick control.. 1998. 2000). parva and T. McKeever et al. these approaches have a number of drawbacks. parva is associated with the extensive lymphoproliferation of schizont-infected cells. Immunization of cattle with a second T... parva sporozoite stabilate in conjunction with long acting tetracylines is used against ECF schizonts (Boulter and Hall. Taracha et al. annulata. 1982. although CD4+ T cells may be involved (Emery et al. The sterile immunity that develops after a primary infection is the basis for live and subunit vaccine development against T.... Initial studies using p67 expressed in E. Jenkins / Veterinary Parasitology 101 (2001) 291–310 301 except that T and B cells (principally CD4+ T cells) rather than monocytes are invaded by sporozoites (see McKeever and Morrison.. Adaptive immunity to T.. 1981. 1999). the host immune response to each and the current control measures against these two species are appreciably different. 1984). Considerable research has also been conducted on the T. 1999). annulata (Musoke et al. Schizont-infected cells are lysed by CD8+ T lymphocytes and killed by macrophages that have been activated by cytokines released by memory CD4+ T cells (Preston et al. Higher levels of protection were achieved by vaccination with SPAG-1 in ISCOMs or mixed with recombinant p67 T. culture-derived T. 2000). parva infection (Honda et al. The innate response to a primary T. 1998). Cattle immunized with two allelic forms of this antigen. While antibodies to T. parva antigen (Boulter et al.. Significant protection was . Boulter and Hall. Preventing sporozoite infection of host cells is the aim of vaccines based on the 67 kDa surface antigen of T. 1998). 1999). annulata infection is carried out by macrophages that produce TNF and NO which suppress parasite proliferation and by NK cells which directly lyse schizont-infected cells (Preston et al. 1997. parva sporozoite challenge (Heussler et al. whereas an “infection and treatment” regimen using a T. The live vaccine against tropical theileriosis is comprised of attenuated.. and T. 1997). 1997). 2000). Vaccine trials against T. Immunization with a mixture of VV expressing p67 and VV expressing IL-2 elicited significant protection in cattle against sporozoite challenge (Honda et al. parva and T. annulata merozoite antigen TAMS-1. parva using recombinant p67 expressed in a variety of delivery vectors have also shown promising results. parva appears to principally involve CD8+ T cell lysis of schizont-infected cells. annulata appears to involve both innate and memory immune responses (Preston et al. annulata piroplasm challenge (d’Oliveira et al. incorporated into ISCOMs showed excellent protection against T.. 1999)... inoculation of cattle with Salmonella dublin expressing recombinant p67 antigen elicited p67-specific humoral and cellular responses and conferred protection against T. 1992. annulata. Acquired immunity to T. 1995).M.

Packham.. Satoskar.E. 1997). Med.. Assoc. The difficulty is to identify the antigens that are targeted by the host’s protective immune response and express and deliver those proteins to induce the appropriate memory response (Watts and Kennedy. H. Evidence of vertical transmission of Neospora sp.E..P. 1997). O’Handley. to achieve solid immunity. 39–47. R. by incorporating antigens from other developmental stages. . but allows for limited parasite replication may be an ideal strategy for protecting susceptible individuals. Biochem.A.L.A. Reynolds. Alberti.P. but not an insurmountable obstacle to developing recombinant protozoan vaccines. The complex nature of host–parasite interactions. Barr. Finlay. Mol.. Vaccine 16. This immunity is the basis of live and attenuated vaccines for several protozoan diseases. Sverlow. L. P. Immunol. Cysteine-rich variant surface proteins of Giardia lamblia. J. J. For many protozoan diseases..302 M. 5. 608–612. M. G. Roberts. Sarmiento..W.. Sierra. A. Specific cellular and humoral immune response in BALB/c mice immunized with an expression genomic library of Trypanosoma cruzi. 210. References Aggarwal. is a formidable.C... J. C. 1998.. Microbiol. Fonte. Taylor for critical reviews of the manuscript. Infante. K. for instance. Am. recombinant antigens should have a longer shelf-life than vaccines composed of live or attenuated organisms.W. Liddell. A. M. In theory. J. 1996. L. Alexander. Nash. As pointed out in a previous review. Neospora infection as a major cause of abortion in California dairy cattle... Parasitol.C. Hoffman.L. typhimurium recombinants expressing TAMS1 antigens (d’Oliveira et al.. but not with S. Conrad. Anderson. 1997. Conrad. 198. J. Vidal. B.. Bluethmann.. Acosta. J. Med.. 1991. the protozoa described above elicit immune responses in most individuals that eventually control parasite replication and limit disease severity.P. Merritt. Rowe... M. P. C.. A. a more feasible goal may be to first develop vaccines that protect against clinical disease (Cox.W. the severity of infection is directly related to the number of replicating parasites in the host. T. Izquierdo. Top. 241–244.. Am. Fachado... C.. 32. Assoc. J. P. Jebbari. Dubey.. A. 1169–1172.. Dubey.. Hidalgo. 1999). A subunit vaccine that protects against clinical signs. J... S. E.D.C. Jenkins / Veterinary Parasitology 101 (2001) 291–310 also achieved by direct immunization with recombinant plasmid DNA pTAMS1. and K. Concluding remarks Although it is difficult to make generalizations about such a diverse group of organisms. 183–195.L. Curr.M. A secondary goal would then be to optimize subunit vaccines.. Anderson. Blanchard. Immunological control of Toxoplasma gondii and appropriate vaccine design.. B.. T.E. Acknowledgements The author wishes to thank Drs. infection in dairy cattle. Barr.F. 1989.. R. H. A. Vet. Many of these “live” vaccines have not gained widespread acceptance because of a requirement for maintaining the cold-chain between production and use in the field. J. 219. Vet..

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