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

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

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

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

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 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

ENDOR spectrum of the CbiY bound flavin radical.

<p>(A) W-band (94 GHz) cw-EPR spectrum of the CbiY flavin radical. Experimental parameters: microwave power 65 nW, modulation frequency 100 kHz, modulation amplitude 1 G, temperature 120 K. Inset is the spectrum of the CbiY flavin radical recorded at X-band EPR (B) W-band Davies pulsed ENDOR spectrum of the CbiY bound flavin semiquinone radical recorded at t = 120 K. The pulse sequence π-T-π/2-τ-π-acq was employed with π = 400ns, T = 20 µs and τ = 1.2 µs. A 18 µs radiofrequency pulsed was applied during T, 1 µs after the initial π pulse. The field was set to g = 2.0039 (33498 G).</p

The <i>bluB</i> gene of <i>Bacillus megaterium</i> encodes DMB synthase.

<p>The aerobic growth of <i>Salmonella enterica</i> strain JE7087, carrying pBAD24 (vector-only, open circles), pBluB9_(Rr) (<i>R. rubrum BluB</i>⁺; black squares), pBluB25_(Bm) (<i>B. megaterium bluB</i>⁺; black triangles), and pCbiY6_(Bm) (<i>B. megaterium cbiY</i>⁺; black diamonds) in NCE minimal medium containing 90 mM ethanolamine, 1 mM MgSO₄, 1× trace minerals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055708#pone.0055708-Balch1" target="_blank">[41]</a>, 50 µg µl⁻¹ ampicillin, and 150 nM dicyanocobinamide. Where indicated, cobinamide was replaced with vitamin B₁₂ (open diamonds), DMB was added at 30 µM (open triangles), and arabinose was added at 250 µM (Panel B). Optical density at 650 nm was measured for 36 h at 37°C. Growth curves were obtained using an ELx808 Ultra Microplate reader (Bio-Tek Instruments). Each growth curve was performed in triplicate, and error bars of one standard deviation are indicated. <b>Panels C and D</b> show aerobic growth of <i>S. enterica</i> strain JE2119 (<i>S. enterica metE205 ara-9 cobC1175</i>::Tn10d[<i>tet</i>⁺]) carrying pBAD24 (vector-only, open circles), pBluB9_(Rr) (<i>R. rubrum bluB</i>⁺; black squares), pBluB25_(Bm) (<i>B. megaterium bluB</i>⁺; black triangles), and pCbiY6_(Bm) (<i>B. megaterium cbiY</i>⁺; black diamonds) in NCE minimal medium containing 11 mM glucose, 1 mM MgSO₄, 1× trace minerals (Balch and Wolfe 1976), 50 µg µl⁻¹ ampicillin, and 15 nM dicyanocobinamide. Where indicated, dicyanocobinamide was replaced with cyanocobalamin (open diamonds), DMB was added at 30 µM (open triangles), and arabinose was added at 500 µM (Panel D). Aerobic growth of JE2119 derivatives took place in 5 ml volumes in 125 ml sidearm flasks which were shaken at 280 rpm at 37°C. Growth was monitored with a Summerson colorimeter (Klett). Each growth curve was performed in triplicate, and error bars of one standard deviation are indicated.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

HPLC analysis of reaction products from <i>in vitro</i> activity assays using BluB_(Rc),

<p><b>BluB_(Bm) and CbiY_(Bm).</b> The reactions were prepared in buffer A at ambient temperature with 100 µM FMN, 8 mM NADH, and 1 mM DTT, and contained either 1.47 mM BluB_(Rc), 1.50 mM BluB_(Bm), or 1.38 mM CbiY_(Bm), respectively. Before HPLC analysis, the reaction mixture was heated to 100°C to denature the enzymes.</p