GTMB 7 - Gene Therapy & Molecular Biology

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Almazán et al: Expressed sequence tags in Ixodes scapularisfeeding was produced in mice vaccinated with a DNApool containing this clone and clone 2F9 of unknownfunction. It is therefore possible that the enhanced feedingeffect on tick larvae was due to clone 2F9 with little or nocontribution of clone 3C12.Microsatellites are a class of genetic markers that arecomposed of STR sequences flanked by unique DNAsequences (Hearne et al, 1992). STRs are highlypolymorphic and widely distributed through the genome.The analysis of tick STRs has been used for identificationof strains of B. microplus (de la Fuente et al, 2000b) andfor the development of a preliminary genetic linkage mapof I. scapularis (Ullman et al, 2003). The STR sequencesdescribed in this study could be used for completion of thegenetic map of I. scapularis as the first step toward thesequencing of this tick genome.Most sequences in the I. scapularis EST data setwere relatively G + C rich, with an average G + C contentof 54%, similar to the 52% reported by Nene et al. (2002)for A. variegatum. The few sequences with a high A + Tcontent probably corresponded to mitochondrial genes,with pathogens) provides new exciting possibilities forscreening and identifying antigens protective against tickinfestations. This approach may also allow foridentification of antigens that interfere with pathogendevelopment and transmission.Recently, Almazán et al. (2003) used cDNA ELIcombined with EST analysis as a rapid method for theidentification of protective antigens against I. scapularisinfestations, demonstrating the role of sequenceinformation in conjunction with new technologies such asbioinformatics and ELI for a systematic andcomprehensive approach to vaccine discovery.One of the advantages of ELI for identification ofprotective antigens is that a priori criteria are notintroduced to direct the selection of candidate genes. Thisapproach, as shown in this study, resulted in potentialvaccine antigens otherwise not predicted, such as clone4F8 that was found to be identical to a nucleotidase.However, nucleotidases are essential for cell growth andthe inhibition of its enzymatic activity would be cytotoxic(Spiegelberg et al, 1999), providing a possible explanationfor their protective properties against tick infestations. TheI. scapularis sequence in clone 4F8 was different from the5’-nucleotidase that was identified and characterizedpreviously by Liyou et al. (1999, 2000) in B. microplus.However, the protective capacity of this protein has notbeen evaluated.As discussed previously by Almazán et al, (2003), apossible explanation for the inhibitory effect on larval tickdevelopment of other vaccine candidates that wereidentified in this study is based on the role that they playin cell growth and maintenance, which is evident forclones identical to beta-adaptin (3E1) and chloride channel(4G11). Beta adaptins are adaptor components required inthe assembly of clathrin-coated plasma membrane pits thatfunction in cell vesicular transport mechanisms includingendocytosis (Camidge and Pearse, 1994; Boehm andBonifacino, 2002), a process actively involved in blooddigestion by ticks and other hematophagous arthropods(Akov, 1982). Chloride channels are also involved in vitalcell functions including the catalysis of counter ioncurrents that accompany primary proton fluxes inendosomal and lysosomal acidification (Koprowski andKubalski, 2001; Iyer et al, 2002). Therefore, interferencewith the process of endocytosis may impair acquisitionand digestion of the tick bloodmeal and result in inhibitionof tick infestations. Another I. scapularis EST (clone3E12) encoded for a protein identical to D. melanogasterclathrin heavy chain, a protein involved in synaptic vesicleendocytosis (Chang et al, 2002). This cDNA is also acandidate protective antigen because it interfers withendocytosis in feeding larvae.The protection capacity of ribosomal and heat shockprotein preparations has been documented previously inother organisms (Elad and Segal, 1995; Silva, 1999;Melby et al, 2000; Cassataro et al, 2002). Recently, Hsp70was demonstrated to be induced in I. ricinus salivaryglands during blood feeding (Leboulle et al, 2002),documenting the role of heat shock proteins inphysiological responses in ticks. Even in the case wheresubstantial homology exists between tick proteins and host(mouse) proteins, analysis of ribosomal proteins suggeststhat differences in the amino acid sequence could directthe host immune response against distinctive, non-selfepitopes, which could be sufficient to induce a protectiveresponse.The results of vaccination and tick infestationdemonstrated that some cDNAs enhance tick feeding. Thiseffect could be due to the expression corroborating thehypothesis that there is a marked difference in codonusage between mitochondrial and nuclear protein codinggenes in the Ixodidae (Nene et al, 2002).Most of the ESTs in our database, although initiallyidentified by ELI of cDNA pools that produced inhibitionof tick infestation, were not characterized further andremain potential candidate antigens for vaccinedevelopment against I. scapularis infestations. Particularlyinteresting were cDNAs that may be involved indevelopmental processes. Clone 4B2, identical to D.melanogaster sequence NP_523710, encoded forcalmodulin, a Ca ++ -binding protein of 149 amino acids thatis involved in fly development. This protein was found tobe expressed in several larval and adult tissues, includingthe larval midgut (Takamatsu et al, 2002). Clone 1C8 hada low degree of identity to D. melanogaster virilizer, agene involved in Sex-lethal (Sxl) splicing and essential forfly male and female viability and embryonic development(Niessen et al, 2001). Clone 2A11 also had a low degree ofidentity to D. melanogaster developmental regulator,Notchless, a key player in the signaling by Notch familyreceptors that are involved in many cell-fate decisionsduring development (Royet et al, 1998). Similarly, clone4A10 had partial identity to the putative homeodomaintranscriptional factor, phtf, a member of a gene family thatplays an important role during development and isconserved between fly, mouse and human (Manuel et al,2000). Other clones with special interest as vaccinecandidates may include those identical to membraneproteins (1D8, 1D11, 3G11) and those putatively involved56
Gene Therapy and Molecular Biology Vol 7, page 57in G-protein-coupled signaling (2B7, 2F12, 4C9). In fact,the clone 3G11 was identical to D. melanogaster BM-40, aprotein of the group of extracellular basement membraneproteins which includes the protective antigen p29 fromHaemaphysalis longicornis (Mulenga et al, 1999).In summary, we have characterized I. scapularis ESTsequences that were selected by cDNA ELI in themouse/tick challenge model because they affected tickdevelopment. Characterization of these ESTs provides abasis for future research on ticks and is a source ofcandidate antigens for use in vaccine developmentdesigned to control tick infestations and/or reducetransmission of pathogens. The combination of ELI withEST appears to be a productive systematic andcomprehensive approach to vaccine discovery.AcknowledgmentsThis research was supported by the project No. 1669of the Oklahoma Agricultural Experiment Station, theEndowed Chair for Food Animal Research (K. M. Kocan,College of Veterinary Medicine, Oklahoma StateUniversity), NIH Centers for Biomedical ResearchExcellence through a subcontract to J. de la Fuente fromthe Oklahoma Medical Research Foundation, and theOklahoma Center for the Advancement of Science andTechnology, Applied Research Grant, AR00(1)-001 andAR02(1)-037. Consuelo Almazán is supported by a grantin-aidfrom the CONACYT, Mexico and an assistantshipfrom the College of Veterinary Medicine, Oklahoma StateUniversity. J. C. Garcia-Garcia is supported by a HowardHughes Medical Institute Predoctoral Fellowship inBiological Sciences. Jerry Bowman is acknowledged forproviding tick larvae. Janet J. Rogers and Sue AnnHudiburg (Core Sequencing Facility, Department ofBiochemistry and Molecular Biology, Noble ResearchCenter, Oklahoma State University) are acknowledged forDNA sequencing and oligonucleotide synthesis,respectively. We thank Joy Yoshioka for editorialassistance.ReferencesAlberti E, Acosta A, Sarmiento ME, Hidalgo C, Vidal T,Fachado A, Fonte L, Izquierdo L, Infante JF, Finlay CM,Sierra G (1998) Specific cellular and humoral immuneresponse in Balb/c mice immunised with an expressiongenomic library of Trypanosoma cruzi. Vaccine 16, 608-612.Almazán C, Kocan KM, Bergman DK, Garcia-Garcia JC, BlouinEF, de la Fuente J (2003) Identification of protective antigensfor the control of Ixodes scapularis infestations using cDNAexpression library immunization. Vaccine 21, 1492-1501.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)Basic local alignment search tool. J Mol Biol 215, 403-410.Altschul, SF, Madden TL, Schäffer AA, Zhang J, Zhang Z,Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms. 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Nature 377, 632-635.Berriz GF, White JV, King OD, Roth FP (2003) GoFish findsgenes with combinations of Gene Ontology attributes.Bioinformatics 19, 788-789.Bior AD, Essenberg RC, Sauer JR (2002) Comparison ofdifferentially expressed genes in the salivary glands of maleticks, Amblyomma americanum and Dermacentor andersoni.Insect Biochem Mol Biol 32, 645-655.Boguski MS, Lowe TM, Tolstoshev CM (1993) dbEST-databasefor "expressed sequence tags". Nat Genet 4, 332-333.Boehm M, Bonifacino JS (2002) Genetic analyses of adaptinfunction from yeast to mammals. Gene 286, 175-186.Brayton KA, Vogel SW, Allsopp BA (1998) Expression libraryimmunization to identify protective antigens from Cowdriaruminantium. Ann N Y Acad Sci 849, 369-371.Camidge DR, Pearse BM (1994) Cloning of Drosophila betaadaptinand its localization on expression in mammaliancells. 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Semin Clin Neuropsychiatry 8,31-36.de la Fuente J, Rodriguez M, Redondo M, Montero C, Garcia-Garcia JC, Mendez L, Serrano E, Valdes M, Enriquez A,Canales M, Ramos E, Boue O, Machado H, Lleonart R, deArmas CA, Rey S, Rodriguez JL, Artiles M, Garcia L (1998)Field studies and cost-effectiveness analysis of vaccinationwith Gavac against the cattle tick Boophilus microplus.Vaccine 16, 366-373.de la Fuente J, Rodriguez M, Montero C, Redondo M, Garcia-Garcia JC, Mendez L, Serrano E, Valdes M, Enriquez A,Canales M, Ramos E, Boue O, Machado H, Lleonart R(1999) Vaccination against ticks (Boophilus spp.): theexperience with the Bm86-based vaccine Gavac. Genet Anal15, 143-148.de la Fuente J, Rodriguez M, Garcia-Garcia JC (2000a)Immunological control of ticks through vaccination withBoophilus microplus gut antigens. Ann N Y Acad Sci 916,617-621.de la Fuente J, Garc_a-Garc_a JC, Gonz_lez DM, Izquierdo G,Ochagavia ME (2000b) Molecular analysis of Boophilus spp.(Acari: Ixodidae) tick strains. 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