Ad2 fiber protein enhances the nasal immunogenicity of BoNT/A β-trefoil in NZW rabbits.

<p>Female NZW rabbits were immunized on days 0, 14 and 28 with the indicated vaccine formulation. Intramuscular immunization with 10 µg of BoNT/A toxoid combined with alum (n = 4) served as a control. BoNT/A Hcβtre (10 µg) was nasally delivered in the absence of adjuvant (n = 5) or combined with CT (2 µg; n = 6) or C48/80 (120 µg; n = 6). BoNT/A Hcβtre-Ad2F (20 µg) was delivered nasally in the absence of adjuvant (n = 5) or combined with CT (2 µg; n = 6) or C48/80 (120 µg; n = 6). BoNT/A Hc (20 µg) was delivered nasally combined with CT (2 µg; n = 3) or C48/80 (120 µg; n = 3). Serum samples collected on day 27 and day 40 were tested for the presence of anti-BoNT/A β-trefoil IgG by ELISA. Serum antibody titers were compared between groups by ANOVA followed by Tukey's multiple comparison test (GraphPad, Prism). <b>a</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80, nasal immunization with Hcβtre-Ad2F, nasal immunization with Hc + CT and nasal immunization with Hc + C48/80. <b>b</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre + CT and nasal immunization with Hcβtre + C48/80. <b>c</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre-Ad2F, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80, nasal immunization with Hc + CT and nasal immunization with Hc + C48/80. <b>d</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, immunization with Hcβtre + C48/80, nasal immunization with Hcβtre-Ad2F and nasal immunization with Hc + C48/80.</p

BoNT/A Hcβtre immunogens induce antibodies that recognize epitopes distinct from those induced by BoNT/A toxoid.

<p>Day 40 sera from a subset of rabbits included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016532#pone-0016532-g001" target="_blank">Figure 1</a> were tested for the presence of antibodies specific for BoNT/A Hc or BoNT/A toxoid by ELISA. <b>a</b>: serum anti-BoNT/A toxoid IgG titers significantly greater than those induced by all other groups. There were no other significant differences between groups.</p

Ad2 fiber protein enhances the induction of BoNT/A-neutralizing antibodies in nasally immunized rabbits.

<p>Female Dutch Belted rabbits (4 per group) were nasally immunized on days 0, 14, 28 and 91 with the indicated vaccine formulation. <b>A</b>. Serum collected on day 105 was tested for the presence of BoNT/A-neutralizing antibodies using a mouse neutralization assay. <b>a</b>: serum BoNT/A-neutralizing activity significantly greater than the neutralizing activity measured in rabbits nasally immunized with Hcβtre, Hcβtre + CT, Hcβtre + C48/80 or Hcβtre-Ad2F; ANOVA and Tukey's multiple comparison, p<0.05. <b>b</b>: serum BoNT/A-neutralizing activity significantly greater than the neutralizing activity measured in rabbits nasally immunized with Hcβtre, Hcβtre + CT, Hcβtre + C48/80 or Hcβtre-Ad2F; ANOVA and Tukey's multiple comparison, p<0.05. <b>B</b>. Hcβtre-Ad2F is superior to Hcβtre for the induction of BoNT/A neutralizing antibodies when delivered with CT or C48/80 as adjuvants. <b>a</b>: p = 0.0006, Mann-Whitney test.</p

Adjuvants enhance the avidity of anti-BoNT/A β-trefoil IgG antibodies.

<p>The relative avidity of anti-BoNT/A IgG serum antibodies was measured using day 162 serum using a modified ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016532#s2" target="_blank">Materials and Methods</a>. <b>A</b>. The percent antibody bound in the ELISA in the presence of 3 M ammonium thiocyanate is indicated for individual serum samples that had measurable anti-β-trefoil IgG. A higher percent antibody bound represents a greater relative avidity for vaccine-induced, antigen-specific antibody. •: represents serum anti-β-trefoil IgG with no detectable BoNT/A neutralizing activity. ○: represents serum anti-β-trefoil IgG with detectable BoNT/A neutralizing activity. <b>B</b>. Average percent antibody bound when samples were organized by the neutralization capacity of the serum. Vaccine-induced BoNT/A neutralizing antibody has significantly greater avidity than non-neutralizing antibody. <b>a</b>: % antibody bound significantly greater than in the no neutralization group. Two-tailed Mann Whitney, p = 0.0239.</p

Schematic representation of Hcβtre-Ad2F fusion protein.

<p><b>A</b>. schematic representation of botulinum neurotoxin type A (heavy and light chains). <b>B</b>. Schematic of β-trefoil domain of BoNT/A heavy chain (Hcβtre). <b>C</b>. Schematic of fusion protein containing β-trefoil domain of BoNT/A heavy chain (Hcβtre) and the adenovirus type 2 fiber protein (Hcβtre-Ad2F).</p

Ad2 fiber protein enhances the nasal immunogenicity of BoNT/A β-trefoil in Dutch Belted rabbits.

<p>Female Dutch Belted rabbits (4 per group) were nasally immunized on days 0, 14, 28 and 91 with the indicated vaccine formulation. Serum collected on days 27, 41 and 105 was tested for the presence of anti-BoNT/A β-trefoil IgG by ELISA. <b>a</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre or nasal immunization with Hcβtre + C48/80. <b>b</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre, nasal immunization with Hcβtre + C48/80 or nasal immunization with Hcβtre-Ad2F. <b>c</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80. <b>d</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre or nasal immunization with Hcβtre + C48/80. <b>e</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre.</p