Determination of Lowest Vaccine Dose for Immune Protection against Ebola Virus Challenge by Adenoviral Vector GP/NP Vaccine

<div><p>(A) Kaplan-Meier survival curve of macaques: Immunization was with rAd vectors expressing GP(Z) and NP(Z) (10⁹–10¹¹, three animals per group; control and 10¹², one animal each), and challenge was performed with the Kikwit strain of Zaire ebolavirus as in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030177#pmed-0030177-g002" target="_blank">Figure 2</a>A. </p> <p>(B) Immune responses in immunized animals. Intracellular flow cytometry was performed to quantify TNF-α production from Ebola-specific CD4⁺ (left graph) or CD8⁺ (right graph) lymphocytes, respectively, from animals immunized as indicated. Immune responses were measured at 3 wk postimmunization. Circles, diamonds, and squares indicate responses for individual animals. Horizontal line indicates the average of individual responses in the immunization group. Results represent the percent cytokine positive in the gated lymphocyte group and background stimulation (DMSO alone) has been subtracted from each sample ( <i>p</i>-values obtained using unpaired Student's <i>t</i>-test; n.s., not significant). </p> <p>(C) Antibody responses in immunized animals. Anti-GP ELISA titers (left graph) and serum neutralizing antibody responses (right graph) were measured as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030177#s2" target="_blank">Methods</a>. </p></div

Elimination of GP Cytopathic Effects and Expression of Transmembrane-Deleted Protein

<div><p>(A) Expression of GP(ΔTM) in HEK293 cells. Ebola GP proteins from supernatants and cell lysates were visualized by SDS-PAGE and Western blot using a polyclonal antibody against Ebola GP.</p> <p>(B) Elimination of cell rounding by GPΔTM. HEK293 cells were transfected with a plasmid that encoded vector control, Ebola GP, or Ebola GPΔTM. Cell monolayers were visualized under phase contrast using a Nikon 40× objective and photographed at 24 h posttransfection.</p></div

Comparative Efficacy of Wild-Type and Point Mutant Glycoprotein Vaccines against Lethal Ebola Virus Challenge

<div><p>(A) Kaplan-Meier survival curve of macaques: Immunization and challenge were performed with the Kikwit strain ebolavirus as in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030177#pmed-0030177-g002" target="_blank">Figure 2</a>A. </p> <p>(B) Immune responses in immunized animals. Left and middle graphs: Intracellular flow cytometry was performed to quantify TNF-α production from Ebola-specific CD4⁺ or CD8⁺ lymphocytes, respectively, from animals immunized as indicated. Immune responses were measured at 3 wk postimmunization. Circles, diamonds, and squares indicate responses for individual animals. Horizontal line indicates average of individual responses in the immunization group. Results represent the percent cytokine positive in the gated lymphocyte group, and background stimulation (DMSO alone) has been subtracted from each sample. ELISA titers of Ebola GP-specific antibodies in sera of vaccinated animals collected at week 3 postimmunization (right graph). ELISA results represent endpoint dilution titers determined by optical density as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030177#s2" target="_blank">Methods</a>. </p></div

Comparative Efficacy of GP and GPΔTM for Protection against Ebola Virus Challenge

<div><p>(A) Kaplan-Meier survival curve of cynomolgus macaques, immunized as indicated, and challenged with 1,000 pfu of Zaire ebolavirus (strain Kikwit) 1 mo post immunization. The <i>x</i>-axis indicates weeks postchallenge. Different immunization groups had <i>n</i> = 3, except for the GP(Z) + NP (10¹²) group ( <i>n</i> = 4), and control ( <i>n</i> = 1). </p> <p>(B) Immune responses in immunized animals. Left and middle graphs: Intracellular flow cytometry was performed to quantify TNF-α production from Ebola-specific CD4⁺ or CD8⁺ lymphocytes, respectively, from animals immunized as indicated (the 10¹² group contains only three CD8 points due to technical error in one sample). Immune responses were measured at 3 wk postimmunization. Circles, diamonds, squares, and triangles indicate responses for individual animals. Horizontal line indicates the average of individual responses in the immunization group. Results represent the percent cytokine-positive in the gated lymphocyte group, and background stimulation (DMSO alone) has been subtracted from each sample. ELISA titers of Ebola GP-specific antibodies in sera of vaccinated animals collected at week 3 postimmunization (right graph). ELISA results represent endpoint dilution titers determined by optical density as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030177#s2" target="_blank">Methods</a>. </p></div

Elimination of GP Cytopathic Effects with Single Point Mutation

<div><p>(A) Expression of point mutants in HEK293 cells. Ebola GP proteins from supernatants and cell lysates were visualized by SDS-PAGE and Western blot using a polyclonal antibody against Ebola GP.</p> <p>(B) Reactivity of point mutants with a conformation-dependent antibody. HEK293 cells were transfected with a control plasmid (red line) or plasmids expressing wild-type (GP[Z], blue line), or mutant (E71D[Z], green line) proteins. 18 h posttransfection, cells were harvested and stained with a GP-specific antibody, and cell surface GP expression was analyzed by flow cytometry.</p> <p>(C) Elimination of cell rounding by amino acid substitution at position 71. HEK293 cells were transfected with a plasmid that encoded vector control, wild-type Ebola glycoprotein from Zaire (GP[Z]) or Sudan/Gulu (GP[S/G]), or their respective point mutations (E71D[Z] and E71D[S/G]). Cell monolayers were visualized under phase contrast using a Nikon 40× objective and photographed at 24 h posttransfection.</p></div