Hapten-specific naïve B cells are biomarkers of vaccine efficacy against drugs of abuse

Abstract

Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address this obstacle, we developed a fluorescent antigen-based enrichment method paired with flow cytometry to characterize hapten-specific B cells. Using this approach, we studied naïve and activated B cells specific for structurally-related model haptens based on derivatization of the morphinan structure at the C6 position on oxycodone or at the C8 position on hydrocodone, and showing different pre-clinical efficacy against the prescription opioid oxycodone. Prior to vaccination, naïve B cells exhibited relatively higher affinity for the more effective C6-derivatized oxycodone-based hapten (6OXY) and the 6OXY-specific naïve B cell population contained a higher number of B cells with greater affinity for free oxycodone. Higher affinity of naïve B cells for hapten or oxycodone reflected greater efficacy of vaccination in blocking oxycodone distribution to brain in mice. Shortly after immunization, activated hapten-specific B cells were detected prior to oxycodone-specific serum antibodies and provided earlier evidence of vaccine failure or success. Analysis of hapten-specific naïve and activated B cells may aid rational vaccine design and provide screening tools to predict vaccine clinical efficacy against drugs of abuse or other small molecules.

Keywords: Addiction; Antibodies; B cell; Prescription opioids; Vaccine.

Figure 1. Vaccine efficacy in preventing oxycodone distribution to the brain in mice

A) target drugs and haptens. B and C) Serum and brain oxycodone concentrations in mice vaccinated with either 6OXY-KLH, 8HYDROC-KLH or unconjugated KLH at 30 minutes after s.c. administration of 2.25 mg/kg oxycodone (n= 6 mice each vaccine group, n= 5 in KLH group). In previous studies, the 6OXY-KLH immunogen was more effective than 8HYDROC-KLH in blocking oxycodone distribution to the brain in rats and preventing oxycodone behavioral effects in both mice and rats [17]. Statistical symbols: * p< 0.05 from unconjugated KLH control or brackets to indicate between group differences.

Figure 2. Hapten-specific B cells enrichment strategy

The whole B cell repertoire, from individual mouse peripheral lymph nodes and spleen, is first incubated with a decoy reagent consisting of PE-AF647 followed by incubation with the hapten-PE (shown 6OXY-PE). All B cells binding the AF647-PE or the hapten-PE detection reagent are positively isolated using anti-PE magnetic beads and magnetic columns. Using flow cytometry, it was possible to identify hapten-specific B cells from PE-specific B cells because the AF647-PE conjugate fluorescences at different wavelength than PE.

Figure 3. B cell gating strategy of 6OXY-specific B cells in a representative dot plot from a mouse vaccinated with 6OXY-KLH

A) singlet gating to remove any cells that have aggregated. B) total lymphocytes. C) total B cells expressing immunoglobulin positive (Ig⁺) but not the following non-B cell markers: CD90.2 (T cells), Gr-1 (neutrophils), CD11.c (dendritic cells) and F4/80 (macrophages). D) To differentiate between B cells that bind 6OXY or 8HYDROC from PE-binding B cells, B cells were further divided using the AF647-PE decoy. E) hapten-specific B cells were further identified as either non antibody-secreting cells (non-ASC) expressing high levels of B220^high or antibody-secreting cells (ASC) expressing high levels of intracellular immunoglobulin (Ig^high). F) hapten-specific B220^high non-ASC B cells were further identified as GL7^high germinal center (GC) or CD38^high naïve/memory (N/M). G) Naïve/memory B cells were further identified as IgM^high or IgM^low/IgD^low switched immunoglobulin (swIg) cells.

Figure 4. Analysis of pre-immunization hapten-specific naïve B cells

To analyze the number of naive B cells specific for morphinan haptens and their affinities for free drug, haptens or immunogens, peripheral lymph nodes and spleen were collected from each mouse and split to perform paired comparison (n= 5-9 mice each group). The numbers of hapten-specific naïve B cells were the sum of B220^high non-ASC and Ig^high ASC B cells. A) Numbers of hapten-specific naïve B cells in samples enriched for 6OXY-PE or 8HYDROC-PE conjugates. B and C) The % recovery of 6OXY- and 8HYDROC-specific naïve B cells after pre-incubation with free oxycodone, haptens and immunogens. Compared to saline pre-incubation control (i.e. 100% recovery of 6OXY-specific B cells), 67% of the enriched 6OXY-specific B cells bound 6OXY-OVA and 47% bound the free oxycodone or the 6OXY free hapten. In contrast, only 10% of recovered 8HYDROC-specific B cells bound free oxycodone and 26% of 8HYDROC-specific B cells bound 1 μM 8HYDROC-OVA. Pre-incubation with 1 μM of either the 8HYDROC-OVA or the control nicotine CMUNic-OVA immunogen decreased by only 10% the recovery of 6OXY-specific B cells suggesting minimal cross-reactivity between B cell subsets specific for 6OXY or 8HYDROC haptens. D) The numbers of 6OXY-specific B cells with high affinity for free oxycodone and 6OXY-OVA were higher than 8HYDROC-specific B cells with high affinity for free oxycodone and 8HYDROC-OVA. Statistical symbols: ** p< 0.01 compared to saline control or brackets to indicate between group differences.

Figure 5. Analysis of hapten-specific B cell numbers shortly after immunization

The number of 6OXY-specific and 8HYDROC-specific B cells were measured at 0, 7 and 14 days after immunization with either 6OXY-KLH, 8HYDROC-KLH or unconjugated KLH. Individual mouse samples were split and each ½ mouse equivalent was enriched with either 6OXY-PE or 8HYDROC-PE. A) Immunization with 6OXY-KLH and 8HYDROC-KLH respectively increased the numbers of 6OXY-specific and 8HYDROC-specific B cells (including B220^high non-ASC and Ig^high ASC) compared to naïve mice and mice immunized with KLH (n= 5-9 mice each group). The punctuated line represents the mean number of hapten-specific B cells in naïve mice. B) The individual numbers of 6OXY- or 8HYDROC-specific activated B cells (B220^high non-ASC and Ig^high ASC) in immunized mice are obtained by subtracting the mean number of hapten-specific naïve B cells from each respective naïve mouse group (represented by the punctuated line in panel A, numbers are the mean from Figure 4, panel A). C) Numbers of 6OXY- and 8HYDROC-specific activated B cell in mice immunized with the lead 6OXY-KLH immunogen. D) Numbers of 6OXY- and 8HYDROC-specific Ig^high ASC B cells in mice immunized with the 6OXY-KLH or the 8HYDROC-KLH immunogen. Statistical symbols: * p< 0.05, ** p< 0.01 from control group or brackets to indicate between group differences.

Figure 6. Analysis of hapten-specific B cells polyclonal responses to vaccination with candidate 6OXY-KLH immunogen

After vaccination with 6OXY-KLH (n= 9 mice) absorbed on Alum, individual samples were split and enriched with 6OXY-PE or 8HYDROC-PE. The 6OXY- and 8HYDROC-specific B cells were composed of: A) GL7^high GC B cells, B) CD38^high N/M B cells, which can be further divided in IgM^high and swIg B cells (shown in C and D); and E) Ig^high ASC B cells. Statistical symbols: * p< 0.05 and ** p< 0.01 compared to naïve mice as shown by brackets.