Babesia bovis:Common Protein Fractions Recognized by OligoclonalB. bovis-Specific CD4+T Cell Lines from Genetically Diverse Cattle

doi:10.1006/expr.1999.4351

Experimental Parasitology

Volume 91, Issue 1, January 1999, Pages 40-51

https://doi.org/10.1006/expr.1999.4351 Get rights and content

Abstract

Stich, R. W., Rice-Ficht, A. C., Tuo, W., and Brown, W. C. 1999.Babesia bovis: Common protein fractions recognized by oligoclonalB. bovis-specific CD4⁺T cell lines from genetically diverse cattle.Experimental Parasitology91,40–51. CD4⁺helper T cells are believed to be important for inducing protective immunity againstBabesia bovisthrough the production of cytokines, including IFN-γ, that will provide help to B lymphocytes for IgG production and activate macrophages to become parasiticidal. To provide maximum protection in an outbred population, an effective vaccine againstB. bovisshould contain antigens that would elicit an IFN-γ response and would be recognized by cattle with diverse genetic backgrounds. To identify potentially protective “universal” T helper (Th) cell antigens, fractions of homogenizedB. bovismerozoites were tested for the ability to stimulate proliferation of oligoclonal CD4⁺, IFN-γ-producing T cell lines derived from four immune animals previously shown to differ in major histocompatibility complex class II expression. HomogenizedB. bovismerozoites were separated by denaturing continuous flow electrophoresis (CFE) on 15, 10, and 7.5% polyacrylamide gels into fractions containing proteins ranging from <14.5 to approximately 95 kDa. Eighteen of 280 CFE fractions elicited anamnestic proliferative responses in all T cell lines tested. Nine of these cross-stimulatory fractions contained proteins of <14.5 to 24.5 kDa, and the remaining ones contained proteins with estimated molecular weights of 30, 31.5, 44.5, 49, 49.5, 54, 62, 72, and 82 kDa. Immunoblot analysis showed that four cross-stimulatory fractions contained a predicted knownB. bovisantigen of similar molecular size. Previous studies had demonstrated that fractionated merozoite proteins stimulatory for CD4⁺Th cell clones had apparent molecular weights similar to those present in 7 of the 18 stimulatory fractions. In the present study, two Th cell clones responded to cross-stimulatory CFE fractions, underscoring the potential to use both oligoclonal and monoclonal Th cell lines to identify commonly recognized polypeptides as potential vaccine antigens.

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The extracellular domain binds to the monomorphic region of MHC class II to increase the affinity of the T cell receptor to the antigen peptide MHC class II complex (Kwong et al., 1990). CD4 + T helper (Th) cells have an essential role in protection against babesiosis (Stich et al., 1999). Destruction of B. bovis infected erythrocytes is mainly carried out by activated splenic macrophages (Brown and Palmer, 1999).
Different allelic forms of bovine CD4 were previously described in cattle and were also observed in Canchim calves examined in the present experiment. However, the functional relevance of these different CD4 phenotypes has not yet been investigated. CD4 + T helper cells are known to play a central role in immune control against Babesia bovis infection. Thus, our study aimed to compare the profiles of immune cells, specific antibody titers and blood infection levels measured by qPCR (quantitative polymerase chain reaction) in calves naturally infected with B. bovis, phenotyped as CD4- (absence of anti-CD4 staining), CD4 + (intermediate staining) or CD4 ++ (high staining). The CD4 mRNA precursor was also measured in these animals. Calves with the CD4- phenotype showed higher amounts of B. bovis DNA in blood samples, compared to the other CD4 phenotypes. It was also observed that these calves with higher levels of infection had lower amounts of natural killer cells and higher expression of the CD4 gene, which can be interpreted as a compensation for the failure of the altered CD4 receptor to recognize relevant B. bovis epitopes.
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Multiple genetically distinct strains of a pathogen circulate and compete for dominance within populations of animal reservoir hosts. Understanding the basis for genotypic strain structure is critical for predicting how pathogens respond to selective pressures and how shifts in pathogen population structure can lead to disease outbreaks. Evidence from related Apicomplexans such as Plasmodium, Toxoplasma, Cryptosporidium and Theileria suggests that various patterns of population dynamics exist, including but not limited to clonal, oligoclonal, panmictic and epidemic genotypic strain structures. In Babesia bovis, genetic diversity of variable merozoite surface antigen (VMSA) genes has been associated with disease outbreaks, including in previously vaccinated animals. However, the extent of VMSA diversity within a defined population in an endemic area has not been examined. We analyzed genotypic diversity and temporal change of MSA-1, a member of the VMSA family, in individual infected animals within a reservoir host population. Twenty-eight distinct MSA-1 genotypes were identified within the herd. All genotypically distinct MSA-1 sequences clustered into three groups based on sequence similarity. Two thirds of the animals tested changed their dominant MSA-1 genotypes during a 6-month period. Five animals within the population contained multiple genotypes. Interestingly, the predominant genotypes within those five animals also changed over the 6-month sampling period, suggesting ongoing transmission or emergence of variant MSA-1 genotypes within the herd. This study demonstrated an unexpected level of diversity for a single copy gene in a haploid genome, and illustrates the dynamic genotype structure of B. bovis within an individual animal in an endemic region. Co-infection with multiple diverse MSA-1 genotypes provides a basis for more extensive genotypic shifts that characterizes outbreak strains.
Human Babesiosis
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Citation Excerpt :
IFN-γ, in synergy with inflammatory cytokines such as tumor necrosis factor-α (TNF-α), activates macrophages to produce nitric oxide that kills intracellular parasites [82,86,87]. Merozoites themselves also can induce inflammatory cytokines and nitric oxide [87,88]. The fine-tuning of IFN-γ and TNF-α by the anti-inflammatory cytokines IL-4 and IL-10 [89] may ensure that the inflammatory reaction remains mainly localized to the spleen.
Human babesiosis is an emerging intraerythrocytic infection caused by protozoal parasites transmitted by ixodid ticks. Babesiosis is endemic in the northeastern and upper midwestern regions of the United States and is found sporadically in other parts of the United States, Europe, Asia, Africa, and South America. Babesial infections range from asymptomatic to severe and occasionally are fatal. Specific laboratory diagnosis of babesial infection is made by morphologic examination of Giemsa-stained blood smears, serology, and amplification of babesial DNA using polymerase chain reaction. The combination of atovaquone and azithromycin is the treatment of choice for mild-to-moderate illness, whereas clindamycin and quinine and exchange transfusion are indicated for severe disease.
High-throughput identification of T-lymphocyte antigens from Anaplasma marginale expressed using in vitro transcription and translation
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The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsial pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. We have used this immunization model to evaluate high-throughput screening of proteins expressed by in vitro transcription and translation (IVTT) for recognition by memory CD4⁺ T-lymphocytes. Fifty selected vaccine candidate antigens identified from the A. marginale genome were expressed from transcriptionally active PCR products using an Escherichia coli-based IVTT system, and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T-lymphocytes from outer membrane immunized animals and evaluated for immunogenicity in proliferation assays. Antigens that consistently stimulated responses were known T-cell antigens major surface protein (MSP)2, MSP3, VirB9, and VirB10 and newly identified T-cell antigens outer membrane protein (OMP)4, OMP9, elongation factor-Tu, Ana29, and OMA87. Specific T-cell stimulation was achieved even at low antigen concentration, and was highly sensitive when compared with unbound IVTT reaction products. This method allows rapid expression and identification of T-lymphocyte antigens for any pathogen for which the genome sequence is available.
Immune control of Babesia bovis infection
2006, Veterinary Parasitology
Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4⁺ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4⁺ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity.
A novel 78-kDa fatty acyl-CoA synthetase (ACS1) of Babesia bovis stimulates memory CD4<sup>+</sup> T lymphocyte responses in B. bovis-immune cattle
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Antigen-specific CD4⁺ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4⁺ T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4⁺ lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1–PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4⁺ T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.

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^☆: The authors thank Sue Ellen Chantler, Debby Alperin, and Daming Zhu for technical assistance and Lisl Shoda and Bill Davis for their advice during the course of this work. This project was supported by National Institutes of Health Grant 2R01-AI30136.

¹: Current address: Department of Veterinary Preventive Medicine, 1900 Coffey Road, Ohio State University, Columbus, OH 43210-1092, U.S.A.

²: To whom correspondence should be addressed. E-mail:[email protected].

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Regular ArticleBabesia bovis:Common Protein Fractions Recognized by OligoclonalB. bovis-Specific CD4+T Cell Lines from Genetically Diverse Cattle☆

Abstract

Experimental Parasitology

Experimental Parasitology

Parasitology Today

Veterinary Parasitology

Cellular Immunology

Molecular and Biochemical Parasitology

Molecular and Biochemical Parasitology

Immunology Letters

Transactions of the Royal Society of Tropical Medicine and Hygiene

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Cellular Immunology

Research in Veterinary Science

Veterinary Parasitology

Prolonged Th1-like response generated by aPlasmodium yoelii

Parasite Immunology

Cell-mediated immune responses toBabesia bovis

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Identification ofBabesia bovisB. bovis

Infection and Immunity

Immunodominant T cell antigens and epitopes ofBabesia bovisBabesia bigemina

Annals of Tropical Medicine and Parasitology

Helper T cell epitopes encoded by theBabesia bigemina rap-1

Infection and Immunity

Babesia bovisB. bovis

Infection and Immunity

Regular Article
Babesia bovis:Common Protein Fractions Recognized by OligoclonalB. bovis-Specific CD4⁺T Cell Lines from Genetically Diverse Cattle☆