Expression and immunogenicity of an 18-residue epitope of HIV1 gp41 inserted in the flagellar protein of a Salmonella live vaccine

• Published in: Research in microbiology 1995, Vol.146 (3), p.203-216
• Main Authors: Newton, S.M.C, Joys, T.M, Anderson, S.A, Kennedy, R.C, Hovi, M.E, Stocker, B.A.D
• Format: Article
• Language: English
• Subjects: AIDS Vaccines - immunology; AIDS/HIV; Animals; Antibody Formation - immunology; Applied microbiology; Bacterial Vaccines - immunology; Biological and medical sciences; Blotting, Western; Epitopes - genetics; Epitopes - immunology; Flagella - ultrastructure; Flagellin - genetics; Flagellin - immunology; Fundamental and applied biological sciences. Psychology; HIV-1 - genetics; HIV-1 - immunology; HIV1, gp41, Epitope, Flagellin, Salmonella; human immunodeficiency virus 1; Humoral immunity, Insertion; In Vitro Techniques; Live attenuated vaccine; Membrane Glycoproteins - genetics; Membrane Glycoproteins - immunology; Mice; Mice, Inbred BALB C; Microbiology; Rabbits; Salmonella - chemistry; Salmonella - genetics; Salmonella - immunology; Salmonella dublin; Vaccin vivant atténué. Immunité humorale, Insertion; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects); Vaccines, Attenuated - immunology; VIH1, gp41, Epitope, Flagelline, Salmonella
• ISSN: 0923-2508; 1769-7123
• DOI: 10.1016/0923-2508(96)80276-2

A synthetic oligonucleotide specifying residues 735–752 of the product of the env gene of HIV1 IIIB was inserted by blunt-end ligation at restriction sites in the hypervariabls, antigenically determinant region IV of two flagellin genes. Its integration, in name and correct orientation, into gene fliC(d) in plasmid pLS408 allowed production of functional flagella when the plasmid was placed in a flagellin-negative aroA live-vaccine Salmonella dublin strain, SL5928. Bacteria thus made motile were immobilized and agglutinated by anti-(735–752 peptide) serum; expression was also shown by immunoelectron-microscopy and by Western blot of whole-cell lysates. Enzyme immunoassay (EIA) of sera of mice given three doses by intraperitoneal injection of the live-vaccine strain producing chimeric flagellin, or of concentrated flagella from it showed production of antibody with affinity for the peptide, and in some sera, also for r-gp160. Pooled serum from mice given five i.p. doses of the live vaccine strain expressing the gp41 epitope at the surface of its flagellar filaments had higher titres of anti-peptide and anti-r-gp160 antibody and weak neutralizing activity on the IIIB isolate (90 % neutralization at 1 100 ). The sera of nine mice given two doses of the live vaccine by the oral route had either no or only very low titres of antibody to flagellar antigen d; they were therefore not tested for anti-peptide activity. Un oligonucléotide synthétique déterminant les résidus 735–752 du produit du gène env du VIH IIIB, a été inséré par ligature des extrémités franches aux sites de restriction de la région IV hypervariable, déterminante antigéniquement, de deux gènes de la flagelline. Son intégration, en disposition et orientation correctes dans le gène fliC(d) du plasmide pLS408 permet la production d'un flagelle fonctionnel quand le plasmide est placé dans la souche vaccinale vivante de Salmonella dublin SL5928 aroA, flagelline-négative. La bactérie rendue ainsi motile est immobilisée et agglutinée par un sérum anti-(peptide 735–752); l'expression du peptide a été aussi mise en évidence par microscopie électronique et par Western blot des lysats de cellules entières. Les dosages immunoenzymatiques des anticorps produits par les sérums des souris auxquelles on a injecté intrapérìtonéalement 3 doses (1) de la souche de vaccin vivant atténué produisant une flagelline chimérique, ou (2) du flagelle concentré de la souche, montrent l'affinité de ces anticorps pour le pepti