Fentanyl conjugate vaccine by injected or mucosal delivery with dmLT or LTA1 adjuvants implicates IgA in protection from drug challenge

Abstract

Fentanyl is a major contributor to the devastating increase in overdose deaths from substance use disorders (SUD). A vaccine targeting fentanyl could be a powerful immunotherapeutic. Here, we evaluated adjuvant and delivery strategies for conjugate antigen vaccination with fentanyl-based haptens. We tested adjuvants derived from the heat-labile toxin of E. coli including dmLT and LTA1 by intramuscular, sublingual or intranasal delivery. Our results show anti-fentanyl serum antibodies and antibody secreting cells in the bone-marrow after vaccination with highest levels observed with an adjuvant (alum, dmLT, or LTA1). Vaccine adjuvanted with LTA1 or dmLT elicited the highest levels of anti-fentanyl antibodies, whereas alum achieved highest levels against the carrier protein. Vaccination with sublingual dmLT or intranasal LTA1 provided the most robust blockade of fentanyl-induced analgesia and CNS penetration correlating strongly to anti-FEN IgA. In conclusion, this study demonstrates dmLT or LTA1 adjuvant as well as mucosal delivery may be attractive strategies for improving the efficacy of vaccines against SUD.

Fig. 1. Immune responses to intramuscular immunization with FEN-BSA are enhanced with dmLT adjuvant.

Groups of Balb/c mice (n = 4) were left naïve or immunized intramuscularly with 8 μg FEN-BSA ± 1 μg dmLT on weeks 0 and 4. Two-weeks later (week 6) serum, spleen, or bone-marrow were collected for antibody evaluations. a Schematic of immunization schedule identifying week of immunizations (IM) and sacrifice for blood, spleen, and bone-marrow sample collections (SAC). b Raw ELISA data for serum anti-BSA or anti-FEN IgG expressed as 405 nm optical density (OD) absorbance. For anti-FEN IgG, plates were coated with FEN-TT. c Compiled ELISA units (EU)/ml of indicated serum anti-BSA and anti-FEN IgG titers assessed by ELISAs graphed on a log2 scale. d Compiled EU/ml of indicated serum anti-FEN IgG1 or IgG2a. e Raw ELISA data for serum anti-FEN IgA and IgM. f Representative images for ASCs IgG/IgA ELISPOT results using splenocytes (Sp) or bone marrow (BM) cells and plates coated with FEN-TT. Blue spots indicate IgG ASCs and red spots IgA ASCs. g Compiled ASC per 10⁶ cells from spleens or bone marrow. Bars at mean + s.e.m. with significance indicated as (***) for P ≤ 0.001 (*) for P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test.

Fig. 2. Immune responses to prime/boost intramuscular immunization with FEN-CRM₁₉₇ are enhanced in magnitude and longevity with dmLT and LTA1 adjuvants.

Groups of Balb/c mice (n = 3–5) were left naïve or immunized IM with 5 μg FEN-CRM ± 150 μg alum, 0.1 μg dmLT, or 5 μg LTA1 on weeks 0 and 3. Two-weeks later (week 5) or six-weeks later (week 9) serum, spleen, and bone-marrow was collected for antibody evaluations. a Schematic of the immunization schedule. b Raw ELISA data for week 5 serum anti-CRM or anti-FEN serum IgG expressed as absorbance. Two anti-FEN ELISA coating antigens were used as indicated by FEN-BSA or FEN-TT. c Compiled week 5 and 9 serum anti-CRM or anti-FEN (coating antigen indicated) serum IgG. d Representative anti-FEN (FEN-TT coating antigen) IgG/IgA ELISPOT images from bone-marrow of week 5 immunized mice. Blue spots indicate IgG and red spots IgA ASCs. e Compiled anti-FEN IgG ASC per 1e6 cells from bone marrow. f Anti-FEN IgG memory B cells per 10⁶ cells differentiated in culture from immunized splenocytes from week 9 post immunization. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3. Immune responses to prime/boost/boost intramuscular FEN-CRM197 immunization are enhanced in magnitude with dmLT and LTA1 adjuvants.

Groups of Balb/c mice (n = 3–5) were left naïve or immunized IM with 5 μg FEN-CRM ± 150 μg alum, 0.1 μg dmLT, or 5 μg LTA1 on weeks 0, 3, and 6. Two-weeks later (week 8) serum and bone-marrow was collected for antibody evaluations. a Schematic of immunization schedule. b Compiled Week 5 and Week 9 serum anti-CRM or anti-FEN (coating antigen indicated) serum IgG. c Compiled EU/ml of indicated serum anti-FEN serum IgG1, or IgG2a ELISAs. d Representative anti-FEN (TT-BSA coated) IgG/IgA ELISPOT images from bone-marrow of week 8 immunized mice. Blue spots indicate IgG and red spots IgA ASCs. e Compiled anti-FEN IgG ASC per 10⁶ cells from bone-marrow. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 4. Immune responses to FEN-CRM₁₉₇ can be generated by sublingual or intranasal booster immunizations containing either dmLT or LTA1 adjuvants.

Groups of Balb/c mice (n = 6) were left naïve or primed by 5 μg FEN-CRM ± 0.1 μg of dmLT or 5 μg of LTA1 by IM delivery. Mice were boosted mucosally on weeks 3 and 6 with 9–10 μg FEN-CRM ± 5 μg dmLT SL or 9 μg FEN-CRM ± 5 μg LTA1 IN, both in 30 μl. On week 8, serum and bone-marrow were collected for immune response evaluation. a Schematic of immunization schedule. b Compiled serum anti-CRM and anti-FEN (FEN-BSA, FEN-TT) serum IgG ELISAs. c Compiled serum anti-FEN (FEN-TT) serum IgG1 or IgG2a ELISAs. d Representative anti-FEN (TT-BSA coated) IgG/IgA ELISPOT images from bone-marrow. Blue spots indicate IgG and red spots IgA ASCs. d Compiled serum anti-FEN (FEN-BSA) IgA ELISA. e Compiled anti-FEN IgG ASC per 10⁶ cells from bone-marrow. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5. Significant correlations between anti-FEN IgG antibody analyses by serum ELISAs and ASC ELISPOT.

a Correlations between indicated IgG ELISAs or ELISPOT results with coating antigen indicated in parenthesis using compiled data from FEN-CRM ± adjuvants IM prime/boost or prime/boost/boost experiments. b Correlations between indicated IgG ELISAs or ELISPOT results with coating antigen indicated in parenthesis using compiled data from FEN-CRM ± adjuvants prime IM and mucosal (SL or IN) booster experiments. c Correlations between indicated IgA ELISAs or ELISPOT results with coating antigen indicated in parenthesis using compiled data from FEN-CRM ± adjuvants prime IM and mucosal (SL or IN) booster experiments. ELISPOT data graphed as bone marrow ASC per 1e6 cells (log10). ELISA data graphed as serum IgG EU/ml (log2). Spearman correlations P values and correlation coefficient (r) indicated on each graph.

Fig. 6. Reduction in fentanyl-induced antinociception and brain tissue distribution in dmLT and LTA1 vaccinated animals compared to alum or antigen alone groups.

Groups of Balb/c mice (n = 5–20) were left naïve or immunized on weeks 0, 3, and 6 with FEN-CRM IM or with adjuvant and route combinations as performed previously. These included 1500 μg alum IM, 0.1–1 μg dmLT IM, 0.1 μg dmLT IM + 5 μg dmLT SL boosters (dmLT SL), or 5 μg LTA1 IM + 5 μg LTA1 IN boosters (LTA1 IN). FEN-CRM was administered at 5 μg for IM delivery and 9–10 μg for mucosal delivery. Blood was collected on week 6, 8, and 10 for serum analyses and mice challenged with fentanyl doses during weeks 9–12. Nociception tests were conducted using tail flick and hotplate assays with tissue taken for drug quantification. a Schematic of immunization schedule. b Compiled serum anti-FEN (FEN-BSA) serum IgG ELISA from week 6, 8, and 10 tailbleeds. c % maximum possible effect (MPE) from tail flick antinociception evaluation. d % MPE from hotplate antinociception evaluation. e Fentanyl concentrations in brain and serum after a final 0.1 mg/kg fentanyl challenge. F Correlation between brain fentanyl levels or tail flick antinociception %MPE vs anti-FEN serum IgG for immunized groups. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001). Spearman correlations P values and correlation coefficient (r) indicated on relevant graph.

Fig. 7. Anti-FEN antibody affinity, IgG2a, and IgA isotypes contributed to protection from fentanyl challenge in dmLT and LTA1 immunized animals.

Serum samples from fentanyl challenge experiments were further characterized for anti-FEN immunity. a IgG antibody binding affinity calculated as % IgG binding to FEN-TT in the presence of a chaotropic agent (week 6). b Compiled serum anti-FEN (FEN-TT) serum IgG1 (week 8), IgG2a (week 8), IgA (week 10) ELISA from tailbleeds. c Ratio of serum anti-FEN (FEN-TT) IgG1/IgG2a. (Zero values for IgG2a were replaced with a 1.) d Correlation between hotplate antinociception vs IgG2a or IgG binding affinity for 0.1 and 1 μg dmLT IM immunization groups only. e Correlation between brain fentanyl levels or tail flick antinociception %MPE vs anti-FEN serum IgA (week 10) for immunized groups. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001). Spearman correlations P values and correlation coefficient (r) indicated on relevant graph.