A lipidated TLR7/8 adjuvant enhances the efficacy of a vaccine against fentanyl in mice

Abstract

Opioid use disorders (OUD) and opioid-related fatal overdoses are a public health concern in the United States. Approximately 100,000 fatal opioid-related overdoses occurred annually from mid-2020 to the present, the majority of which involved fentanyl or fentanyl analogs. Vaccines have been proposed as a therapeutic and prophylactic strategy to offer selective and long-lasting protection against accidental or deliberate exposure to fentanyl and closely related analogs. To support the development of a clinically viable anti-opioid vaccine suitable for human use, the incorporation of adjuvants will be required to elicit high titers of high-affinity circulating antibodies specific to the target opioid. Here we demonstrate that the addition of a synthetic TLR7/8 agonist, INI-4001, but not a synthetic TLR4 agonist, INI-2002, to a candidate conjugate vaccine consisting of a fentanyl-based hapten, F₁, conjugated to the diphtheria cross-reactive material (CRM), significantly increased generation of high-affinity F₁-specific antibody concentrations, and reduced drug distribution to the brain after fentanyl administration in mice.

Fig. 1. INI-4001 is a synthetic TLR7/8 agonist.

a Chemical structure of INI-4001. b, c SEAP activation in HEK293 cells expressing human TLR7 (b) or human TLR8 (c) by indicated doses of INI-4001. Data were reported as fold change in SEAP production over vehicle control and shown as mean ± SEM. d, e Human PBMCs from five different donors were stimulated with indicated doses of INI-4001 for 24 h. Supernatants were then analyzed for IFNα (d) and TNFα (e) production by ELISA.

Fig. 2. INI-2002 is a synthetic TLR4 agonist.

a Chemical structure of INI-2002. b SEAP activation in HEK293 cells expressing human TLR4 by indicated doses of INI-2002. Data were reported as fold changes in SEAP production over vehicle control. c–f Human PBMCs from five different donors were stimulated with indicated doses of INI-2002 for 24 h. Supernatants were then analyzed for indicated cytokine production using a custom U-PLEX MesoScale Discovery (MSD) assay for all cytokines shown.

Fig. 3. Adjuvanting F₁-CRM with INI-4001 plus alum preferentially increases anti-F₁ IgG2a antibody concentration.

Mice were vaccinated twice, IM, with 5 µg F₁-CRM plus 7.68, 9, or 22.5 µg alum, 0.1, 1, or 10 µg INI-2002, 0.1, 1, or 10 µg INI-4001, the combination of INI-2002 + alum, or the combination of INI-4001 + alum as indicated. Fourteen days after the second vaccination, blood was collected, and anti-F₁ IgG (a, b), IgG1 (c, d), and IgG2a (e, f) antibody concentrations were measured by ELISA. Antibody responses to vaccines adjuvanted with INI-2002 are shown on the left (a, c, e). Antibody responses to vaccines adjuvanted with INI-4001 are shown on the right (b, d, f). Statistical analysis was conducted by one-way ANOVA with Fisher’s LSD for multiple comparisons (GraphPad Prism). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; color of asterisks indicates comparison group.

Fig. 4. The addition of INI-2002 to INI-4001 plus alum does not further increase F1-specific antibody concentrations.

Mice were vaccinated twice, IM, with 5 µg F1-CRM plus 9, 22.5, or 24 µg alum, 1 µg INI-2002, 10 µg INI-4001, the combination of INI-2002 + INI-4001, INI-2002 + alum, INI-4001 + alum, or INI-2002 + INI-4001 + alum as indicated. Fourteen days after the second vaccination, blood was collected and anti-F₁ IgG (a), IgG1 (b), and IgG2a (c) antibody concentrations were measured by ELISA. Statistical analysis was conducted by one-way ANOVA with Fisher’s LSD for multiple comparisons (GraphPad Prism). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; color of asterisks indicates comparison group.

Fig. 5. The activation of TLR4 by INI-2002 is blocked by the addition of F₁, CRM, or F₁-CRM.

HEK cells expressing human TLR4 were stimulated with F₁, CRM, F₁-CRM, INI-2002, or the combination of F₁, CRM, or F₁-CRM plus INI-2002. The order of addition of F₁, CRM, or F₁-CRM and INI-2002 varied when both were used as indicated in the table by either “first” or “second”. The addition of the second component occurred 1 h after the addition of the first component. INI-4001 and vehicle only were included as negative controls. Statistical analysis was conducted by one-way ANOVA with Fisher’s LSD for multiple comparisons (GraphPad Prism). **p ≤ 0.01 and ****p ≤ 0.0001. Orange asterisks and text indicate a comparison to INI-2002, and black asterisks and text indicate a comparison to vehicle only.

Fig. 6. Addition of INI-4001 increases antibody avidity to F₁.

a Mouse serum samples from Fig. 3 were used to measure the dissociation rate (K_diss) between F₁ and polyclonal mouse serum for each individual serum sample using the Octet Red 96e instrument (Fortebio). Dissociation rate constants (K_diss) were calculated by processing raw data using ForteBio HT analysis software version 11.1.3.50. All data were inspected for quality of fit to the calculated curve (R² > 0.95), the response between 0.25–3 (nm shift), and residual value <10% of the maximum response fitted to the curve. b Average K_diss per group from (a) was plotted against average anti-F₁ IgG antibody concentration as shown in Fig. 3a. Correlation was calculated via nonlinear regression analysis where both axes are graphed on a log scale. Statistical analysis was conducted by one-way ANOVA with Fisher’s LSD for multiple comparisons (GraphPad Prism). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; color of asterisks indicates comparison group.

Fig. 7. INI-4001 plus alum increases the efficacy of F₁-CRM to protect mice against fentanyl challenge.

Mice were immunized, IM, on days 0, 14, and 28, with indicated vaccine components, followed by drug challenge with 0.05 mg/kg fentanyl s.c. on day 35. a Fentanyl-induced antinociception on a hotplate, measured as percent maximum possible effect (%MPE), b fentanyl-induced bradycardia, measured as heart rate percent change from baseline, c serum fentanyl concentration, and d brain fentanyl concentration. Data were mean ± SEM. Sample size: n = 6 per group. Statistical analysis was conducted by one-way ANOVA with Tukey’s multiple comparisons post hoc test (GraphPad Prism). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 compared to CRM-only vaccinated control or as noted by bars.