The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice
<p>Size-exclusion chromatography (SE-HPLC) chromatograms for NIC7 conjugates made with high and low ratios of sulfo <span class="html-italic">N</span>-hydroxysuccinimide (sNHS) and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC). The top panel shows the SE-HPLC profile for a conjugate produced with a sNHS/EDC ratio of 0.14. For this sample, the amount of Main 1 vs. Main 2 is almost 1:1. The middle panel shows the SE-HPLC profile for a conjugate produced with a sNHS/EDC ratio of 0.86 and Main 2 is present to a much lower extent. The bottom panel shows a control sample of unconjugated CRM<sub>197</sub> showing that Main 2 is not present.</p> "> Figure 2
<p>Analytical ultracentrifugation (AUC) analysis of Main 1 vs. Main 2. Fractions from SE chromatography were collected and analyzed by AUC. Fractions corresponding to Main 1 (dashed line) and Main 2 (dotted line) show significantly different sedimentation coefficients. The black trace is from a sample of the load material for the SE-HPLC run.</p> "> Figure 3
<p>The effect of varying sNHS/EDC ratios on Main 1, Main 2, and hapten load. The amounts of Main 1 and Main 2 for conjugates at each ratio were determined by integrating the area under the peaks from SE-HPLC. Main 1 is represented by the light gray columns, and Main 2 by the dark gray columns. The hapten load for each sample as determined by acid hydrolysis is shown by the curve.</p> "> Figure 4
<p>Effect of different sNHS/EDC ratios on antibody (Ab) titers. BALB/c mice (<span class="html-italic">n</span> = 10/group) were immunized on Days 0 and 21 by IM injection with 10 µg of different NIC7-CRM conjugates adjuvanted with alum (10 µg Al<sup>3+</sup>) + CpG (20 µg). Plasma was collected on Day 20 and 28 and anti-nicotine antibody levels determined by ELISA. Anti-nicotine Ab titers post-Dose 1 and 2 are shown in Panels (<b>A</b>) and (<b>B</b>), respectively. Statistical significance was calculated by a one-factor analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 5
<p>The effect of different sNHS/EDC ratios on Ab function. BALB/c mice (<span class="html-italic">n</span> = 10/group) were immunized on Days 0 and 21 by IM injection with 10 µg of NIC7-CRM conjugates adjuvanted with alum (10 µg Al<sup>3+</sup>) + CpG (20 µg). On Day 28, animals received an IV injection of <sup>3</sup>H-nicotine (0.05 mg/kg) and plasma and brains collected. Panel (<b>A</b>) shows % reduction of nicotine levels in brain (ng-eq/g), and Panel (<b>B</b>) shows % increase of nicotine levels in plasma (ng-eq/mL) compared to control non-immunized mice. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; **** <span class="html-italic">p</span> < 0.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of NIC7 Conjugates Using Varied Concentrations of sNHS
2.2. Hapten Load by Acid Hydrolysis
2.3. Size-Exclusion Chromatography (SE-HPLC)
2.4. Non-Reduced Capillary Gel Electrophoresis (CGE)
2.5. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS)
2.6. Analytical Ultracentrifugation (AUC)
2.7. Adjuvants
2.8. Animal Procedures
2.9. Anti-Nicotine Ab Titers
2.10. In Vivo Functional Assays
2.11. Statistical Analysis
3. Results
3.1. Effect of Varying sNHS/EDC Ratios on Main 1, Main 2, and Hapten Load
3.2. The Effect of Different sNHS/EDC Ratios on Anti-Nicotine Responses
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Molar Ratio sNHS:EDC | SE-HPLC | CGE %HMMS | Hapten Load | |
---|---|---|---|---|
%Main 2 | %Main 1 | |||
0.14 | 47.1 | 48.2 | 2.3 | 13 |
0.27 | 47.6 | 46.4 | 2.3 | 14 |
0.41 | 42.3 | 49.6 | 3.0 | 13 |
0.70 | 29.9 | 59.6 | 1.8 | 12 |
0.78 | 24.8 | 64.6 | 2.0 | 11 |
0.86 | 25.0 | 63.9 | 2.6 | 11 |
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Thorn, J.M.; Bhattacharya, K.; Crutcher, R.; Sperry, J.; Isele, C.; Kelly, B.; Yates, L.; Zobel, J.; Zhang, N.; Davis, H.L.; et al. The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice. Vaccines 2017, 5, 11. https://doi.org/10.3390/vaccines5020011
Thorn JM, Bhattacharya K, Crutcher R, Sperry J, Isele C, Kelly B, Yates L, Zobel J, Zhang N, Davis HL, et al. The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice. Vaccines. 2017; 5(2):11. https://doi.org/10.3390/vaccines5020011
Chicago/Turabian StyleThorn, Jennifer M., Keshab Bhattacharya, Renata Crutcher, Justin Sperry, Colleen Isele, Barbara Kelly, Libbey Yates, James Zobel, Ningli Zhang, Heather L. Davis, and et al. 2017. "The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice" Vaccines 5, no. 2: 11. https://doi.org/10.3390/vaccines5020011