title: Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli.
abstract: BACKGROUND
  Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB) when fused to p24.
  
  
  RESULTS
  A fusion between ricin toxin B subunit and p24 HIV (RTB/p24) was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin.
  
  
  CONCLUSIONS
  In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when conjugated to an HIV structural protein. This is the first report in which a eukaryotic toxin produced in E. coli is employed as an adjuvant to elicit immune responses to p24 HIV core antigen.
authors.affiliation: Centro de Investigación y de Estudios Avanzados (CINVESTAV), Unidad Irapuato, Km 9.6 Libramiento Norte, 36500 Carretera Irapuato-León, Irapuato, Guanajuato, México. albulb@rocketmail.com
authors.forename: Alberto J
authors.initials: AJ
authors.lastname: Donayre-Torres
authors.forename: Ernesto
authors.initials: E
authors.lastname: Esquivel-Soto
authors.forename: María de Lourdes
authors.initials: Mde L
authors.lastname: Gutiérrez-Xicoténcatl
authors.forename: Fernando R
authors.initials: FR
authors.lastname: Esquivel-Guadarrama
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Gómez-Lim
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D016655:HIV Core Protein p24 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D015658:HIV Infections / Q000276:immunology* / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012276:Ricin / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000276:Adjuvants, Immunologic; D000914:Antibodies, Viral; D016655:HIV Core Protein p24; D011993:Recombinant Fusion Proteins; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D012276:Ricin