RESUMEN
Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.
Asunto(s)
Antígenos de Protozoos/inmunología , Babesia/inmunología , Babesiosis/veterinaria , Linfocitos T CD4-Positivos/inmunología , Enfermedades de los Bovinos/inmunología , Memoria Inmunológica , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Babesia/genética , Babesia bovis/genética , Babesia bovis/inmunología , Babesiosis/inmunología , Babesiosis/parasitología , Southern Blotting , Bovinos , Enfermedades de los Bovinos/parasitología , Clonación Molecular , Secuencia Conservada , Cristalinas/genética , Proteínas de Choque Térmico/genética , Sueros Inmunes/inmunología , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.
Asunto(s)
Babesia bovis/fisiología , Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Orgánulos/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Babesia bovis/genética , Babesia bovis/inmunología , Babesia bovis/metabolismo , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Orgánulos/inmunología , Pruebas de Precipitina , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Análisis de Secuencia de ADNRESUMEN
Despite convincing evidence that T cells are critical for both cellular and humoral immunity against haemoprotozoan parasites, the difficulty of performing meaningful experiments in cattle that would define the role of T cells in immunity to Babesia spp. has impeded research in this area. However, experiments performed ex vivo with immune T cells can reflect in-vivo events, and provide valuable insight into the nature of immunogenic proteins and the responding lymphocytes. The progress made towards identification of the immunogenic proteins and epitopes that stimulate anamnestic CD4+ type-1 (interferon-gamma-producing) T-cell responses in cattle immune to challenge with Babesia bovis or B. bigemina is the subject of the present review.
Asunto(s)
Antígenos de Protozoos/análisis , Babesia bovis/inmunología , Babesia/inmunología , Linfocitos T/inmunología , Animales , Babesiosis/inmunología , Antígenos CD4/inmunología , Bovinos , Epítopos/análisis , Inmunoglobulina G/inmunología , Activación de Macrófagos , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Rhoptry-associated protein-1 (RAP-1) homologues of Babesia bigemina and Babesia bovis are promising candidates for inclusion in subunit vaccines against these hemoprotozoan parasites. Partial protection against challenge infection has been achieved with native forms of these antigens, but the mechanism of immunity has not been thoroughly defined. We previously demonstrated that a panel of antigen-specific T helper cell clones derived from B. bigemina RAP-1-immunized cattle expressed relatively high levels of interferon-gamma (IFN-gamma) protein and transcript and low levels of interleukin-4 (IL-4), indicative of a type 1 immune response. In the current study we present evidence that subcutaneous immunization with native B. bigemina RAP-1 protein in RIBI adjuvant induces a predominant type 1 immune response in vivo, characterized by relatively high levels of IFN-gamma and IL-2 and low levels of IL-4 and IL-10 mRNA in the draining prescapular lymph node. Ex vivo restimulation of draining lymph node lymphocytes with specific antigen resulted in proliferation and enhanced expression of IL-2 and IFN-gamma, whereas IL-4 and IL-10 transcript levels remained relatively low. These findings show that our previously described cytokine profiles of antigen-specific cloned T cell lines are representative of autologous in vivo responses and confirm that type 1 recall responses to B. bigemina RAP-1 can be evoked in immunized animals by native parasite antigen.
Asunto(s)
Antígenos de Protozoos/inmunología , Citocinas/biosíntesis , Inmunización , Proteínas Protozoarias/inmunología , Animales , Bovinos , División Celular/inmunología , Células Clonales , Femenino , Interferón gamma/biosíntesis , Interleucina-4/genética , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Transcripción GenéticaRESUMEN
The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.
Asunto(s)
Antígenos de Protozoos/inmunología , Babesiosis/inmunología , Enfermedades de los Bovinos/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos de Protozoos/clasificación , Antígenos de Protozoos/genética , Babesia bovis/clasificación , Babesiosis/epidemiología , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/epidemiología , Células Clonales , Secuencia Conservada , Citocinas/biosíntesis , Epítopos/inmunología , Eritrocitos/parasitología , Inmunidad , Epítopos Inmunodominantes , Activación de Linfocitos , Datos de Secuencia Molecular , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.
Asunto(s)
Antígenos de Protozoos/inmunología , Babesia/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Secuencia de Bases , Bovinos , Línea Celular , Citocinas/genética , Inmunización , Activación de Linfocitos , Datos de Secuencia Molecular , Vacunas Antiprotozoos/inmunologíaRESUMEN
Human recombinant interleukin-10 (IL-10) was previously shown to inhibit accessory cell (AC)-dependent proliferation of bovine parasite-specific T helper 1 (Th1), Th2, and Th0 cells in an IL-2-reversible manner (Brown, W.C., Woods, V.M., Chitko-McKown, C.G., Hash, S.M., and Rice-Ficht, A.C., 1994. Infect. Immun. 62, 4697-4708). The present study was therefore designed to determine whether the effect of IL-10 on T cell proliferation corresponded with downregulated expression of cytokines, or their receptors, important for T cell growth. The effects of IL-10 on cellular proliferation and expression of IL-2, IL-4, IL-2 receptor (IL-2R; p55), and IFN-gamma by Babesia bovis- or Fasciola hepatica-specific Th cell clones were simultaneously evaluated. As shown previously, IL-10 strongly inhibited proliferation of all types of Th cell clones, although this did not correspond with reduced expression of IL-2 or IL-4 mRNA or their products. In contrast, expression of IL-2R mRNA was consistently reduced in the IL-10-treated clones. These results indicate that IL-10 does not inhibit AC-dependent proliferation of bovine Th cells by downregulating T cell cytokines; rather, IL-10 may act by downregulating IL-2R p55 expression and subsequent signal transduction leading to decreased cellular proliferation. IFN-gamma production was also consistently downregulated in the presence of IL-10.
Asunto(s)
Babesiosis/tratamiento farmacológico , Enfermedades de los Bovinos/tratamiento farmacológico , Fascioliasis/tratamiento farmacológico , Interferón gamma/biosíntesis , Interleucinas/farmacología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Animales , Babesiosis/metabolismo , Babesiosis/patología , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/patología , Enfermedad Crónica , Células Clonales , Regulación hacia Abajo , Fascioliasis/metabolismo , Fascioliasis/patología , Interleucina-10/farmacología , Interleucinas/biosíntesis , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Receptores de Interleucina-2/química , Proteínas Recombinantes/farmacología , Linfocitos T Colaboradores-Inductores/parasitologíaRESUMEN
The trypomastigote specific surface antigens of Trypanosoma cruzi are encoded by a supergene family which includes the TSA family. The TSA family is characterized by the presence of a 27-bp tandem repeat array in the coding region. Here, we report the characterization and analysis of the three TSA family members in the Esmeraldo strain of the parasite. In this strain 2 distinct telomeric members are expressed abundantly as 3.7-kb mRNAs, while the remaining member is located at an internal chromosomal site and is expressed at less than 2% of the level seen for the telomeric members. Based on hybridization to DNA separated by PFGE, 3 chromosomes of sizes 1.8 Mb, 0.98 Mb, and 0.90 Mb each contain one of the telomeric members. In addition, the two smaller chromosomes also contain the single internal member. Since both chromosomes contain similar TSA family members, and vary only slightly in size, we suggest that they are homologues. Comparisons of the nucleotide sequences of the different members of the family show that the internal gene differs from the telomeric genes primarily in sequences found 3' of the repeat array. These comparisons also reveal that the three genes are analogous, supporting the hypothesis that short segments between the family members are exchanged by gene conversion events. We propose that similar conversion events between members of different gene families may generate some of the diversity found within the supergene family.
Asunto(s)
Antígenos de Protozoos/genética , Genes Protozoarios , Familia de Multigenes , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/genética , Secuencia de Bases , Evolución Biológica , ADN Protozoario/genética , Conversión Génica , Expresión Génica , Variación Genética , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Trypanosoma cruzi/crecimiento & desarrollo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaAsunto(s)
Genes Protozoarios , Glicoproteínas , Neuraminidasa/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Datos de Secuencia Molecular , Familia de Multigenes , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Trypanosoma cruzi/inmunologíaRESUMEN
In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes.
Asunto(s)
Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Transcripción Genética , Trypanosoma cruzi/genética , Glicoproteínas Variantes de Superficie de Trypanosoma , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Secuencia de Bases , Northern Blotting , Southern Blotting , Endodesoxirribonucleasas/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes , Hibridación de Ácido Nucleico , Poli A/genética , ARN/genética , ARN Mensajero , ARN Protozoario/genética , Homología de Secuencia de Ácido Nucleico , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/metabolismoRESUMEN
A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.