title: Fusion protein vectors to increase protein production and evaluate the immunogenicity of genetic vaccines.
abstract: Genetic immunization is a method for vaccination and laboratory antibody production where antigen-expressing plasmids are introduced into animals to elicit immune responses. Although genetic immunization works well for many antigens, problems can arise with protein sequences that (i) are toxic to host cells, (ii) are difficult to translate by mammalian cells, or (iii) evade immune presentation. We demonstrate here the ability to increase protein production and antigen secretion by the simple method of fusing poorly expressed sequences to well-expressed heterologous proteins. Proof-of-principle is demonstrated here using the poorly translated HIV-1 envelope whose protein production is rescued by fusing this antigen to the carboxy-termini of two well-expressed proteins: the cytoplasmic green fluorescent protein and the secreted human protein a1-antitrypsin. This approach represents a simple and substantially less expensive method to increase protein and antigen production than codon-optimization strategies. It may therefore be more useful than whole gene codon replacement to enable inexpensive laboratory antibody production of poorly expressed antigens and for large-scale genomic protein or antigen screening efforts. Finally, we demonstrate a second benefit of this antigen fusion strategy in which the test antigen is "sandwiched" between two positive control antigens. By this approach, we demonstrate the intrinsic lack of immunogenicity of HIV-1 envelope under conditions when robust antibody responses are generated against its fusion protein partners, but not against this evasive antigen. These fusion protein vectors therefore represent a simple approach to not only increase antigen production, but also assess antigen production and immunogenicity in vivo.
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, 77030, USA.
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: M A
authors.initials: MA
authors.lastname: Barry
mesh_terms: D002460:Cell Line; D005069:Evaluation Studies as Topic; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics*; D000515:alpha 1-Antitrypsin / Q000235:genetics
chemical_list: D015699:HIV Envelope Protein gp120; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D000515:alpha 1-Antitrypsin; D049452:Green Fluorescent Proteins