Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice.
The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.
Andrés
Isabel
I
Unidad de Microbiología, Facultad de Farmacia, Univ de Valencia, Avda, Vicente Andrés Estelles s/n. 46100-Burjassot, Spain.
Rodríguez-Díaz
Jesús
J
Buesa
Javier
J
Zueco
Jesús
J
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D002473:Cell Wall; D005260:Female; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008813:Mice, Inbred ICR; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics / Q000276:immunology*; D012400:Rotavirus Infections / Q000276:immunology / Q000517:prevention & control; D022243:Rotavirus Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000914:Antibodies, Viral; C105281:CCW14 protein, S cerevisiae; D008562:Membrane Glycoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D022243:Rotavirus Vaccines; D029701:Saccharomyces cerevisiae Proteins; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus