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. 2009 Jun 2;27(27):3544-52.
doi: 10.1016/j.vaccine.2009.03.069. Epub 2009 Apr 14.

The mast cell activator compound 48/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen

Afton L McGowen  1 , Laura P Hale, Christopher P Shelburne, Soman N Abraham, Herman F Staats
Affiliations

The mast cell activator compound 48/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen

Afton L McGowen et al. Vaccine. .

Abstract

We evaluated the safety and efficacy of the mast cell activator compound 48/80 (C48/80) when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA+C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin neutralizing antibodies. C48/80 induced a balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection site neutrophil influx within 24h. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses.

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Figures

Figure 1
Figure 1
Serum anti-rPA IgG geometric mean titers after intradermal immunization in the ear pinna with 0.5 μg rPA with or without adjuvant on days 0 and +21. Serum samples taken on day +42 were tested by ELISA. Bars represent the geometric mean titers for each group on day +42 for all replicates, with error bars representing the 95% confidence level (CL). 0.1 μg CT, 1.0 μg CT, 1.0 μg CpG, and 10 μg CpG groups have an n = 5. 3.0 μg C48/80 group n = 10. rPA alone, 10 μg C48/80, and 30 μg C48/80 groups have an n = 15. * p < 0.05 over rPA alone and rPA plus 3 μg C48/80. ** p < 0.01 over rPA plus 10 μg C48/80.
Figure 2
Figure 2
Serum anti-rPA IgG1, IgG2a, IgG2b, and IgG3 geometric mean titers after intradermal immunization in the ear pinna with 0.5 μg rPA in PBS with or without adjuvant on days 0 and +21. Bars represent the geometric mean titers for each group on day +42 for all replicates, with error bars representing the 95% CL. 1.0 μg CT has an n = 3. 0.1 μg CT, 1 μg CpG, and 10 μg CpG have an n = 5. 3.0 μg C48/80 group n = 10. rPA alone, 10 μg C48/80, and 30 μg C48/80 groups have an n = 15. Serum samples were tested by ELISA. 2 p < 0.05 over IgG2a; 3 p < 0.05 over IgG2b; *IgG3 was significantly lower than all other subclasses (p < 0.01).
Figure 3
Figure 3
Serum lethal toxin neutralization titer 75 on day +42 after intradermal immunization in the ear pinna with 0.5 μg rPA with or without adjuvant on days 0 and +21. Bars represent the geometric mean titers for each group on day +42 for all replicates, with error bars representing the 95% CL. rPA alone n = 12. 10 μg C48/80 and 30 μg C48/80 groups have an n = 15. 0.1 μg CT, 1.0 μg CT, 1 μg CpG, and 10 μg CpG groups have an n = 5. Error bars represent 95% CL. Groups with neutralization titers below 1:128 were assigned a value of 2 for graphical representation and statistical analysis. * p < 0.05 over rPA alone. ** p < 0.05 over rPA alone and rPA plus 10 μg C48/80. *** p < 0.001 over rPA alone and rPA plus 10 μg C48/80.
Figure 4
Figure 4
Serum anti-rPA IgE geometric mean titers after intradermal immunization in the ear pinna with 0.5 μg rPA in PBS with or without adjuvant on days 0 and +21. Lines represent the geometric mean titers for each group on day +42 for all replicates. Serum samples were tested by ELISA. The Y axis is set at the threshold of detection for this assay, 16. 0.1 and 1.0 μg CT groups have an n = 5 and n = 4, respectively. All mice vaccinated with CT had detectable IgE. rPA alone mice had no detectable IgE. 1 of 15 mice vaccinated with 10 μg C48/80 had detectable IgE, while 8 of 15 mice vaccinated with 30 μg C48/80 had detectable levels. 1.0 and 10 μg CpG induced IgE in 2 of 5 and 2 of 4 mice, respectively. ** p < 0.01 over rPA alone and rPA plus 10 μg C48/80.
Figure 5
Figure 5
Ear swelling in mm 24 hours after intradermal immunization in the ear pinna with 0.5 μg rPA in PBS with or without adjuvant on day 0. Bars represent the mean swelling for each group for all replicates. 1.0 μg CT and 10 μg CpG groups have an n = 5. rPA alone and 30 μg C48/80 groups have an n = 15. Measurements were made using a dial thickness gauge. Error bars represent standard deviation. * p < 0.01 over rPA alone. ** p < 0.001 over rPA alone. *** p < 0.001 over all other groups.
Figure 6
Figure 6
Histological changes in the ear pinna following intradermal vaccination. Mice were immunized with 0.5 mg rPA in PBS with or without the indicated adjuvant. Hematoxylin and eosin- stained sections are shown 4 hours (A-E) or 24 hrs (F-J) after immunization. Stars indicate areas of prominent edema and white arrows indicate inflammatory infiltrates. Toluidine Blue-stained sections (K-O) show that mast cells (black arrows) are readily detected following immunization with PA alone or with CT or CpG adjuvants, but not following immunization with c48/80.
Figure 7
Figure 7
Total number of mast cells present per mm2 in ear sections taken at 4 (a) or 24 (b) hours after intradermal vaccination in the ear pinnae. Sections were stained with Toluidine Blue and mast cells were counted in a minimum of 14 (0.16mm2) fields at 40×. Error bars represent standard deviation. * p < 0.05 against all other groups.
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