T and B cell responses following immunization.

<p>B10.BR mice were vaccinated I.M. with Ad-CMVZGP (1×10⁵, 1×10⁶ and 1×10⁷ IFU/mouse) or Ad-CAGoptZGP (1×10⁴, 1×10⁵ and 1×10⁶ IFU/mouse) and splenocytes were harvested 8 days later for A. IFN-γ CD8+ T cells frequency analysis or B. Neutralizing antibody (NAB) titers. Four to five mice were analyzed per group and the experiment was repeated twice. Levels of NAB to ZEBOV-EGFP were evaluated 25 days post-vaccination. Error bars represent the standard deviation of the data. n.d. refers to assays that were not done for Ad-CAGoptZGP at that IFU/mouse. * p = 0.0454; ** p = 0.1.</p

Oral Vaccination with PEGylated Adenovirus Improves the B-cell Mediated but Not the T-cell Mediated Immune Response Against Ebola Glycoprotein.

<p>Naïve mice and those with pre-existing immunity were vaccinated with 1×10¹⁰ particles of either unmodified (Vaccine) or PEGylated (PEG-Vaccine) Ad5-ZGP by oral gavage. Pre-existing immunity (PEI) was induced by I.M. injection with 5×10¹⁰ particles of adenovirus expressing beta-galactosidase (AdlacZ). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). (C) Profile of anti-Ebola-specific IgG antibodies. (D) Profile of anti-Ebola-specific IgA antibodies. In all panels, error bars represent the standard deviation of the data.</p

T cell frequency analysis at day 6 post-immunization.

<p>Groups of 4 B10.BR mice were vaccinated I.M. with 1×10⁸ IFU/mouse of Ad-CMVZGP or Ad-CAGoptZGP and splenocytes were harvested 6 days later, re-stimulated with the peptide TELRTFSI, and production of IFN-γ, TNF-α, and Il-2 from CD8+ T cells was monitored by FACS. Error bars represent the standard deviation of the data. The experiment was repeated once and showed similar results. * p<0.001; ** p<0.001; *** p<0.01; **** p<0.01.</p

Intranasal Vaccination Induces Anti-Ebola Specific Ig Isotypes in Bronchioalveolar Lavage Fluid in the Absence and Presence of Pre-Existing Immunity.

<p>Forty-five days after immunization, bronchioalveolar lavage fluid was analyzed for Ebola-specific IgG (panel A) and IgA (panel B) antibodies by ELISA. The optical densities obtained from each treatment group are presented to serve as a measure of relative concentration. PEI - pre-existing immunity to adenovirus serotype 5.</p

Intranasal Immunization Induces Production of Neutralizing Antibodies in the Lung in the Presence of Pre-Existing Immunity.

<p>Forty-five days after immunization, bronchioalveloar fluid was analyzed for the presence of neutralizing antibody by measuring the reduction in infectious titer of ZEBOV-EGFP. The reciprocal dilution plotted for each treatment group (n = 10) reflects the dilution at which the ability of the ZEBOV-EGFP vector to infect target cells was reduced by >50%. Data represent average values obtained from each treatment group and error bars represent the standard deviation of the data. NOTE - Data grouped on the left side of the figure under the label “Serum” is reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003548#pone-0003548-g001" target="_blank">Figure 1B</a> of this manuscript and is included here for comparison. PEI - pre-existing immunity to adenovirus serotype 5.</p

Intranasal Immunization with Recombinant Adenovirus Expressing Ebola Glycoprotein Affords Protection Against Lethal Challenge Even in the Presence of Pre-Existing Immunity.

<p>Naïve mice (n = 10) were vaccinated with a single dose of 1×10¹⁰ particles of adenovirus expressing the Ebola glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×10¹⁰ particles of adenovirus 5 expressing beta-galactosidase (AdlacZ) were also vaccinated in the same manner. Twenty-eight days later, mice were challenged with 200 LD₅₀ of mouse-adapted Ebola virus (Zaire strain). Data represent survival (panel A) and loss of body weight (panel B) over time and is reported as average body weight from each treatment group. Mock - age matched, untreated, unchallenged mice included in this data set to indicate normal weight variation over time. NOTE: Data for naïve mice immunized by the oral route (P.O.) and those with pre-existing immunity and vaccinated by the I.M. route (I.M.+PEI) were not included in this figure for visual clarity. All naive mice immunized orally survived challenge while none in the I.M.+PEI group survived.</p

Pre-Existing Immunity Against Adenovirus Does Not Compromise the Systemic or Mucosal Cellular Response Generated Against Ebola Glycoprotein After Intranasal Immunization.

<p>The frequency of IFN-γ positive mononuclear cells was analyzed from the spleen (panels A and C), the lung via bronchioalveolar lavage (BAL) and the intestine via the mesenteric lymph nodes (MLN) and Peyer's patches (PP) (panels B and D) 45 days post-immunization by ELISPOT. Samples were obtained from naïve animals (Panels A and B) and those with pre-existing immunity to adenovirus serotype 5 (Panels C and D). Cells were plated at 1×10⁵ or 1×10⁴ cells per well, stimulated with the TELRTFSI peptide and expression of IFN-γ detected with an anti-mouse IFN-γ antibody. Cells isolated from BAL, MLN or PP of four mice were pooled and samples tested in duplicate. In each panel, the number of spot-forming cells (SFC) per million mononuclear cells (MNCs) is shown on the y-axis. Please note - the scale of this axis differs between panels to accent the differences between treatment groups. Error bars represent the standard deviation of the data. Animals immunized with an irrelevant adenoviral vector (AdlacZ) served as negative controls. Positive results obtained from this group are indicative of artificial cellular stimulation that may have occurred during processing and culturing of samples.</p

Survival and weight loss of mice challenged with ZEBOV 28 days post-vaccination.

<p>Eight to nine B10.BR mice were used in each group.</p>1<p>not applicable.</p

Western blot expression analysis of Ad-CMVZGP or Ad-CAGoptZGP.

<p>Proteins isolated from infected HEK 293 cells were separated by a 10% SDS PAGE then transferred to PVDF membrane. A mouse monoclonal anti-ZGP was used as the primary antibody and a goat anti-mouse horseradish peroxidase (HRP) conjugated antibody as the secondary antibody. 24, 48 and 72 hours indicate the time of total protein harvest post-infection. Band density corresponding to each lane is shown as determined by densitometry of the bands. The control represents untreated HEK 293 cells. An M.O.I. of 5 was used to infect the cells with either Ad-CMVZGP or Ad-CAGoptZGP for each time point. The preparation of Ad-CAGoptZGP or Ad-CMVZGP used had a non-infectious to infectious ratio of 73∶1or 19∶1 respectively.</p

Pre-Existing Immunity Against Adenovirus Does not Compromise the Strength of the Cellular and Humoral Immune Response Against Ebola Glycoprotein After Intranasal Immunization.

<p>Naïve mice (n = 10) were vaccinated with a single dose of 1×10¹⁰ particles of adenovirus expressing the Ebola Zaire glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×10¹⁰ particles of adenovirus serotype 5 expressing beta-galactosidase (AdlacZ) 30 days prior to vaccination were also immunized in the same manner. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1∶320. Animals given a single dose of (AdlacZ) served as negative controls (AdlacZ Control). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). The TELRTFSI peptide, specific for the Ebola Zaire glycoprotein (0.4 µg/well), was incubated with 1×10⁶ splenocytes and cells analyzed by flow cytometry. (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). In both panels, error bars represent the standard deviation of the data.</p