Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins

Abstract

Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)₄] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)₄-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development.

Keywords: carrier protein; conjugate vaccine; hapten; heroin; linkers; oxycodone.

Figure 1.

Effect of hapten design and linker chemistry on vaccine efficacy against oxycodone in mice. (A) Structure of oxycodone and oxycodone-based haptens. BALB/c mice (n ≥ 7/group) were immunized with unconjugated KLH control, OXY(Gly)₄-KLH, OXY(PEG)₃-KLH, or OXY(Gly)₄-S-KLH adsorbed on alum on days 0, 14, and 28. Vaccine efficacy was evaluated on day 35. (B) Oxycodone-specific serum IgG antibody titers. Oxycodone concentrations in (C) serum and (D) brain at 30 min after a 2.25 mg/kg sc oxycodone challenge. Data are mean ± SEM. Statistical symbols: * represents experimental groups compared to the KLH-only negative control, and # represents between group comparisons. ** or ##, p ≤ 0.01, and **** or ####, p ≤ 0.0001, using two-tailed unpaired t tests with Welch’s correction (B, C, D) and a log-transformed, one-way ANOVA paired with Tukey’s multiple comparison test (C, D).

Figure 2.

Vaccine efficacy requires intact T cell signaling. Serum was collected on day 35, and oxycodone-specific IgG titers were analyzed via ELISA after the following immunizations. (A) C57BL/6 WT and TCRα^−/− mice (n = 5/group) were sc immunized with OXY(Gly)₄-KLH adsorbed on alum on days 0, 14, and 28. (B) BALB/c mice (n = 5/group) were sc immunized with OXY(Gly)₄-KLH, OXY-Ficoll, or OXY-Dextran adsorbed on alum on days 0, 14, and 28. p value symbols: * represents individual experimental groups compared to the control. *, p ≤ 0.05, and ***, p ≤ 0.001, using two-tailed unpaired t tests with Welch’s correction (A and B). Data are presented as mean ± SEM.

Figure 3.

Vaccines formulated with different carrier proteins show equivalent efficacy against oxycodone and expansion of early hapten-specific B cell subsets. BALB/c mice (n ≥ 4/group) were sc immunized with unconjugated KLH, OXY-KLH, OXY-EcoCRM, OXY-rTTHc, OXY-CRM₁₉₇, or OXY-TT, adsorbed on alum adjuvant. (A) Oxycodone-specific serum IgG titers. (B) Serum and (C) brain concentrations of oxycodone after administration of 2.25 mg/kg oxycodone. (D) Oxycodone-specific serum IgG titers vs brain oxycodone concentration (from individual data in panels A and C). BALB/c mice (n ≥ 4/group) were IM immunized with unconjugated KLH, OXY-KLH, OXY-EcoCRM, OXY-rTTHc, or OXY-sKLH (GMP-grade subunit KLH), formulated with alum. At 7 days post-immunization, B cell lymphocytes from the spleen and lymph nodes were analyzed by flow cytometry. OXY-specific B cells are expressed as percentage (%) of the total lymphocyte population after magnetic enrichment. Shown are OXY-specific B cell subsets: (E) antibody-secreting plasma B cells and (F) germinal center (GC) B cells. p value symbols: * represents experimental groups compared to the KLH-only negative control, and # represents interexperimental group comparisons. * or #, p ≤ 0.05, ** or ##, p ≤ 0.01, ***, p ≤ 0.001, and ****, p ≤ 0.0001, by a log transformed (A), one-way ANOVA paired with Tukey’s multiple comparison test (A, B, C), linear regression (D), and a log transformed, two-tailed unpaired t tests with Welch’s correction (E, F). Line and error bars represent mean ± SEM.

Figure 4.

Characterization of carrier protein and conjugates by MALDI-TOF. The EcoCRM, rTTHc, CRM₁₉₇, and TT carrier proteins were analyzed before and after conjugation to the OXY(Gly)₄ hapten. Upper panels (A, B, C, and D) show carrier proteins prior to conjugation, and lower panels (E, F, G, and H) show results after conjugation. Haptenation ratio (HR) was calculated as follows: (MW_{OXY(Gly)4–rTTHc} – MW_rTTHc)/MW_OXY(Gly)4. Samples were analyzed using an Applied Biosystems/MDS SCIEX 54800 MALDI TOF/TOF analyzer operated in high-mass linear mode.

Figure 5.

Characterization of carrier proteins and conjugates by DLS. The carrier protein (75 μL) was assessed before and after conjugation. The percent intensity of (A) EcoCRM, (B) rTTHc, (C) CRM₁₉₇, (D) TT, (E) native KLH, and their respective OXY-containing conjugates are shown. Panels F–J show the percent volume of each carrier protein and their respective conjugates. Dashed lines represent the carrier protein alone. Solid lines represent the carrier proteins conjugated to the OXY(Gly)₄ hapten. The Z average [particle diameter (d) in nm] is expressed as a decadic logarithm. Data were analyzed with a 633 nm laser under an 173-degree backscatter.

Figure 6.

Characterization of carrier proteins and conjugates by SEC-HPLC and SDS-PAGE. Size exclusion chromatography analysis performed on (A) EcoCRM (62.3 kDa), (B) rTTHc (38.8 kDa), (C) OXY-EcoCRM, and (D) OXY-rTTHc. (E) Image of precast 3–8% polyacrylamide gel. From left to right, in the order of molecular weight reference markers, rTTHc, OXY-rTTHc, EcoCRM, and OXY-EcoCRM.

Figure 7.

In vivo and in vitro characterization of heroin vaccines containing different carriers. BALB/c mice (n ≥ 5/group) were IM immunized with either unconjugated sKLH plus EcoCRM (control), M-sKLH, or M-EcoCRM adsorbed on alum on days 0, 14, and 28. (A) Morphine-specific serum IgG antibody titers. At 30 min after a 1 mg/kg heroin challenge, cumulative concentration of heroin, 6-AM, and morphine in (B) serum and (C) brain. (D) Heroin-induced antinociception in the hot plate assay. Data are mean ± SEM. Statistical symbols: * represents experimental groups compared to the negative control (sKLH+EcoCRM). *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, and ****, p ≤ 0.0001 using one-way ANOVA with Tukey’s multiple comparison test. DLS characterization of (E) M-sKLH and (F) M-EcoCRM. Dashed lines represent the unconjugated carrier protein, and solid lines represent the heroin conjugate vaccines. The Z average [particle diameter (d) in nm] is expressed as log base 10. Data were analyzed with a 633 nm laser under a 173-degree backscatter.