Article Figures & Data
Figures
- Fig 1
Timeline for administration of virus-antibody complexes (VACs), sample collection, necropsy, and assessment of the immune response for naïve mice (A) and those with preexisting immunity (PEI) (B). Mice were given a single dose of 1 × 1011 virus particles mixed with different concentrations of anti-adenovirus antibodies (naïve mice) or 28 days after establishment of preexisting immunity by tail vein injection in a volume of 100 μl. Control mice were given 100 μl of sterile PBS and gentamicin in the same manner. Six hours after treatment, blood was collected from the saphenous vein and serum processed for assessment of cytokines, platelets, and serum transaminases. Blood was also collected on days 3 and 7 for assessment of serum transaminases and platelets. Mice from each group (n = 4) were sacrificed on days 4 and 7, and portions of liver and spleen were cryopreserved for sectioning and histochemical detection of beta-galactosidase. Remaining tissue and other organs (heart, kidney, lung, lymph nodes) were harvested and snap-frozen for assessment of virus distribution by real-time PCR. Ten days after treatment, mice (n = 5) were sacrificed and splenocytes processed for assessment of T cell responses. Tissue samples were also taken at this time point for assessment of virus distribution patterns. On day 28, blood was collected from the saphenous vein of remaining animals for characterization of anti-beta-galactosidase and anti-adenovirus antibodies. These animals (5 per group) were sacrificed on day 42 and splenocytes processed for assessment of immunological memory by CFSE staining and flow cytometry. †, terminal bleed and necropsy of animals at the denoted time point.
- Fig 2
Characterization of stock serum by ELISA isotyping (A) and Western blotting (B). (A) Isotyping assays revealed that the antibody stock used to create antibody-virus complexes mostly consisted of anti-adenovirus IgG antibodies with Ig2b being the primary isotype in the 500-, 5-, and 0.05-ND50 preparations. Marked amounts of IgM antibodies were detected in the 500-ND50 preparation only. Values for isotypes present in the 0.005- and 0.0005-ND50 preparations fell below the detection limit of the assay. Error bars indicate the standard deviations from the average results of 4 separate preparations for each serum concentration. (B) Western blot of adenovirus capsid proteins resolved on a 9% SDS-PAGE gel. Each lane was incubated with a different dilution of stock serum. Lane 1, 5 ND50; lane 2, 0.5 ND50; lane 3, 0.05 ND50; lane 4, 0.005 ND50; lane 5, 0.0005 ND50. Serum concentrations at or above 5 ND50 bound the primary adenovirus capsid proteins (hexon, penton, fiber). Fiber binding was lost at the 0.5-ND50 concentration. Hexon binding was detected in the 0.05- and 0.005-ND50 preparations. Binding of adenovirus capsid proteins could not be detected at the 0.0005-ND50 concentration.
- Fig 3
VACs elicit limited transgene expression after systemic administration. First-generation adenoviral vectors were mixed with various concentrations of mouse anti-adenovirus antibodies and given to C57BL/6 mice by tail vein injection (1 × 1011 particles/ml). Animals were sacrificed and livers were evaluated for beta-galactosidase expression by histochemical staining 6 h (second column), 4 days (third column), and 7 days (fourth column) after administration. Samples obtained from the spleen were also evaluated for transgene expression at the 6-h time point (first column). Transgene expression could not be detected in any samples from mice given the 500-ND50 preparation (E to H). Similar results were found for the 5-ND50 preparation (data not shown). Transgene expression could be detected in sections obtained from animals given the 0.05-ND50 preparation (I to L), and the level was significantly lower than that for mice given the virus alone at all time points (A to D).
- Fig 4
VACs at a concentration of 0.05 ND50 can elicit strong T-cell mediated and humoral immune responses against an encoded transgene. (A) CTL responses elicited by VACs. Splenocytes harvested 10 days after treatment were restimulated in vitro for 5 days and tested for specific lysis on MC57 target cells infected with adenovirus expressing beta-galactosidase in a 6-h 51Cr release assay. Percent specific lysis is expressed as a function of different effector-to-target ratios (6:1, 12.5:1, 25:1, 50:1, and 100:1). (B) Anti-beta-galactosidase neutralizing antibody profile after a single dose of VACs in C57BL/6 mice. Serum was analyzed 28 days after treatment for the presence of antibody to beta-galactosidase. Fifty percent endpoint titers were calculated according to the method of Reed and Muench. (C) Anti-adenovirus neutralizing antibody levels. The presence of neutralizing antibody against the adenoviral vector was determined by assessing the ability of collected sera to block infection of HeLa cells by virus expressing beta-galactosidase. The 50% endpoint titer is plotted according to treatment. (D) Memory response. Splenocytes were isolated 42 days after treatment, stained with CFSE, and stimulated with beta-galactosidase-specific peptide for 5 days. Cells positive for CD8+, CD44hi, and CD62Llo were evaluated for CFSE staining by flow cytometry. Data represent the degree of effector CD8 T cell expansion after stimulation. Data illustrated in each panel reflect the means and standard deviations of results for five animals per group. Statistical significance was determined between individual treatment groups and vehicle controls by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *, P ≤ 0.05; **, P ≤ 0.01.
- Fig 5
VACs significantly reduce virus-induced toxicity. (A) Serum cytokine levels. IL-6 and IL-12 (p70) were assessed 6 h after systemic administration of a single dose of either virus alone (1 × 1011 virus particles), the same dose of virus mixed with different amounts of anti-adenovirus antibody, or saline (PBS). (B) Platelets. Fourteen days before treatment, baseline platelet counts were determined (t = 0). A notable drop in platelet count was observed only in animals given virus alone. (C) Kinetic profile of serum alanine aminotransferase (ALT). Normal levels for C57BL/6 mice fall within the range of 24 to 140 U/liter (40). (D) Kinetic profile of serum aspartate aminotransferase (AST). Normal levels for C57BL/6 mice fall within the range of 72 to 288 U/liter (40). In each panel, data reflect average values ± the standard error of the mean for 5 mice from each treatment group. Statistical significance was determined between individual treatment groups and vehicle controls by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *, P ≤ 0.05; **, P ≤ 0.01.
- Fig 6
Anti-adenovirus antibody at a concentration of 0.05 ND50 facilitates strong effector memory responses against an encoded transgene in naive mice. (A) Frequency of IFN-γ-secreting CD8+ T cells. Naïve mice were given either virus alone (1 × 1011 virus particles), the same amount of virus mixed with different concentrations of anti-adenovirus antibody, or saline (PBS, negative control) systemically. Ten days later, 1 × 106 splenocytes from each animal were incubated with a beta-galactosidase-specific peptide and responsive cells analyzed by flow cytometry. (B) Anti-beta-galactosidase neutralizing antibody profile. Serum was analyzed 28 days after treatment by ELISA. Fifty percent endpoint titers were calculated according to the method of Reed and Muench. (C) Anti-adenovirus neutralizing antibody. NAB titers were determined by assessing the ability of collected sera to block infection of HeLa cells by adenovirus expressing beta-galactosidase. The 50% endpoint titer is plotted according to treatment. (D) Memory response. Splenocytes were isolated 42 days after treatment, stained with CFSE, and stimulated with beta-galactosidase-specific peptide. CD8+ cells also positive for CD44hi and CD62Llo were evaluated for CFSE staining by flow cytometry. Data illustrated in each panel reflects the means and standard errors of the means for five animals per group. Statistical significance was determined between individual treatment groups by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *, P ≤ 0.05; **, P ≤ 0.01.
- Fig 7
Preexisting immunity to adenovirus serotype 5 does not significantly compromise the immune response elicited by some VACs. Preexisting immunity was established by intramuscular injection of 1 × 1011 particles of AdEGFP 28 days prior to administration of VACs. At that time, mice had a circulating anti-adenovirus NAB titer of a 184.2 ± 32.4 reciprocal dilution. Animals given saline served as negative controls (PBS). (A) Frequency of IFN-γ-secreting CD8+ T cells. Ten days after treatment, 1 × 106 splenocytes from each animal were harvested and incubated with a beta-galactosidase-specific peptide. Responsive cells were quantitated by flow cytometry. (B) Anti-beta-galactosidase antibody profile after administration of VACs. Serum was analyzed 28 days after treatment. Fifty percent endpoint titers are plotted according to treatment and were calculated according to the method of Reed and Muench. (C) Anti-adenovirus neutralizing antibody. Neutralizing antibody titers were determined by assessing the ability of sera to block infection of HeLa cells by unmodified virus expressing beta-galactosidase. The 50% endpoint titer is plotted according to treatment. (D) Memory response. Cells positive for CD8+, CD44hi, and CD62Llo were evaluated for CFSE staining by flow cytometry. Data represent the degree of effector CD8+ T cell expansion after stimulation for each treatment group. Data illustrated in each panel reflect the means and standard errors of the means for five animals/group. Statistical significance was determined between individual treatment groups and vehicle controls or between naïve animals and those with preexisting immunity to adenovirus by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *, P ≤ 0.05; **, P ≤ 0.01. PEI, preexisting immunity to adenovirus.
- Fig 8
VACs at a 0.05-ND50 ratio significantly reduce the cytokine response and virus-induced thromobcytopenia in mice with PEI. (A) IL-6 secretion. IL-6 was assessed in serum collected 6 h after systemic administration of either virus alone (AdlacZ), virus mixed with different concentrations of anti-adenovirus antibody, or saline (PBS). (B) IL-12 secretion. IL-12 (p70) was also measured 6 h after treatment from a minimum of five mice per group. (C) Serum alanine aminotransferase (ALT). ALT was measured at the time when significant increases are commonly noted in mice given virus alone, 7 days after treatment. Normal levels for C57BL/6 mice are 24 to 140 U/liter (40). (D) Serum aspartate aminotransferase (AST). AST was also measured at the time when significant increases are commonly noted in mice given virus alone, the 7-day time point. AST levels normally fall within the 72- to 288-U/liter range (40). (E) Kinetic profile of platelet counts for naïve mice. Fourteen days before treatment, baseline platelet counts were determined (t = 0). Notable thrombocytopenia was detected in animals given virus alone and the 0.0005-ND50 preparation. (F) Kinetic profile of platelet counts for mice with PEI. Platelet counts were significantly reduced in mice 3 days after virus to establish PEI was given, with levels returning to baseline prior to administration of VACs 28 days later (data not shown). In each panel, statistical significance was determined either between individual treatment groups and vehicle controls or between naïve animals and those with preexisting immunity to adenovirus by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *, P ≤ 0.05; **, P ≤ 0.01.
Tables
- Table 1
Infectious titer as determined by two in vitro assays for each production lot of virus-antibody complexesa
Treatment ITA (ivp/ml) PLA (PFU/ml) PFU:IVP ratio Adenovirus alone 4.08 ± 0.22 × 1010 4.5 ± 0.48 × 1010 1:1.1 Virus + 500 ND50 1.02 ± 0.52 × 108 3.2 ± 0.95 × 106 1:32 Virus + 5 ND50 1.60 ± 0.55 × 108 3.6 ± 0.28 × 106 1:44 Virus + 0.05 ND50 2.30 ± 0.92 × 108 4.2 ± 0.73 × 106 1:55 Virus + 0.005 ND50 2.53 ± 0.22 × 1010 NA Virus + 0.0005 ND50 3.62 ± 0.15 × 1010 NA ↵a The input for each of these preparations was 1.0 × 1011 virus particles/ml, as determined by measuring the absorbance of each preparation at 260 nm. ITA, infectious titer assay; ivp, infectious virus particles; PLA, plaque assay; NA, not assayed.
