The frequency of naive and early-activated hapten-specific B cell subsets dictates the efficacy of a therapeutic vaccine against prescription opioid abuse

Abstract

Translation of therapeutic vaccines for addiction, cancer, or other chronic noncommunicable diseases has been slow because only a small subset of immunized subjects achieved effective Ab levels. We hypothesize that individual variability in the number of naive and early-activated hapten-specific B cells determines postvaccination serum Ab levels and vaccine efficacy. Using a model vaccine against the highly abused prescription opioid oxycodone, the polyclonal B cell population specific for an oxycodone-based hapten (6OXY) was analyzed by flow cytometry paired with Ag-based magnetic enrichment. A higher frequency of 6OXY-specific B cells in either spleen biopsies or blood, before and after immunization, correlated to subsequent greater oxycodone-specific serum Ab titers and their efficacy in blocking oxycodone distribution to the brain and oxycodone-induced behavior in mice. The magnitude of 6OXY-specific B cell activation and vaccine efficacy was tightly correlated to the size of the CD4(+) T cell population. The frequency of enriched 6OXY-specific B cells was consistent across various mouse tissues. These data provide novel evidence that variations in the frequency of naive or early-activated vaccine-specific B and T cells can account for individual responses to vaccines and may predict the clinical efficacy of a therapeutic vaccine.

Figure 1. Study design and evaluation of vaccine efficacy

A) Analysis of 6OXY-specific B cells was performed either in spleen biopsy or blood samples before and after immunization with 6OXY-KLH or KLH in alum on days 0, 14 and 28 (n=12 each group). B-E) Shown data from a BALB/c mice cohort that received partial splenectomy a week after the third immunization. On day 35, 2.25 mg/kg s.c. oxycodone was administered to mice for testing vaccine's effect on oxycodone distribution to serum and to the brain, and on oxycodone nociception. B) 6OXY-specific serum IgG titers in mice immunized with 6OXY-KLH or unconjugated KLH. Effect of immunization on: oxycodone distribution to C) serum and D) the brain, and E) oxycodone antinociception. The maximum possible effect (MPE) represents hind paw lifts on a hot plate (25). *p<0.05 and ***p<0.001; brackets indicate group differences.

Figure 2. Splenic polyclonal hapten-specific B cells before and after immunization

6OXY-specific B cells were analyzed by spleen biopsy prior to immunization (n=24), 14 days after the first immunization and 7 days after the last immunization (n=12 mice each group). A) Representative flow cytometry plots of 6OXY-specific B cells in naïve mice, or mice immunized with either 6OXY-KLH or KLH. Data shown were collected on day 35. To analyze 6OXY-specific B cells, B cells bound to either (6OXY)_n-PE or PE-AF674 were positively isolated by magnetic enrichment (23). Using flow cytometry, B cells bound to (6OXY)_n-PE were distinguished and analyzed for B cell-specific markers (23). 6OXY-specific B cells were either B220^high non-antibody secreting cells or Ig^high ASC B cells. 6OXY-specific B220^high B cells were either CD38^low GL7^high GC or CD38^high naïve and memory B cells. CD38^high GL7^- B cells were further identified as IgM^high or IgM^neg and IgD^neg swIg B cells (20; 23). 6OXY-specific B cells were B) ASC, C) GC, D) swIg B, and E) IgM^high naive and memory. Data shown are the number of 6OXY-specific B cells per spleen biopsy fragments. Data are mean±SEM. Each group is a composite of three independent experiments for a total n=24 before immunization and n=12 each group after immunization. *p<0.05, **p<0.01, and ***p<0.001 compared to KLH group; ### p<0.001 compared to naïve mice.

Figure 3. Number of splenic hapten-specific B cells prior to immunization correlated to vaccine efficacy

Pre-vaccination B cell analysis by spleen biopsy was performed in a naïve cohort of mice and then randomly immunized with either KLH or 6OXY-KLH (n=12 each group). Data shown are the number of 6OXY-specific B cells per spleen biopsy sample from individual mice. In mice immunized with 6OXY-KLH: A) 6OXY-specific serum IgG titers vs serum oxycodone, B) 6OXY-specific IgM^high B cells, and C) 6OXY-specific swIg B cells vs 6OXY-specific serum IgG titers. In mice immunized with 6OXY-KLH or KLH: D) 6OXY-specific serum IgG titers vs brain oxycodone, E) 6OXY-specific IgM^high B cells and F) 6OXY-specific swIg B cells vs brain oxycodone. Each group is a composite of three independent experiments with n=12 each immunization group.

Figure 4. Early-activated splenic 6OXY-specific B cells correlated with vaccine efficacy

6OXY-KLH elicited GC and ASC B cells 14 days after immunization. Mice were immunized on days 0, 14 and 28, and challenged with 2.25 mg/kg oxycodone a week after the third immunization. Distribution of 6OXY-specific B cell subsets in spleen biopsies from individual mice: A) before, and B) after immunization. Immunization with 6OXY-KLH increased: C) the ratio of 6OXY-specific (CD38^-) to (CD38⁺) GL7⁺ B cells over time, and D) serum oxycodone concentrations. At 14 days post-vaccination: E) early GC activation, shown as the ratio of 6OXY-specific (CD38^-) to (CD38^-) GL7⁺ B cells, and F) 6OXY-specific ASC B cells, correlated to greater vaccine efficacy on serum oxycodone at day 35. Data shown are the number of 6OXY-specific B cells per spleen biopsy sample from individual mice. Data are mean±SEM. Data included three independent experiments with a total n=12 each group. **p<0.01, and ***p<0.001 compared to KLH, ### p<0.001 compared to naïve mice; brackets indicate group differences.

Figure 5. Frequency of hapten-specific B cells in blood correlates to vaccine efficacy

Using the (6OXY)₄-SA-PE and (control)₄-SA-PE-AF reagents and magnetic enrichment, B cells were analyzed in 200μl of blood collected before and after immunization with either 6OXY-KLH or KLH. A) Immunization reduced distribution of oxycodone to the brain, and B) early serum IgG titers correlated to subsequent blockage of oxycodone distribution to the brain. C) The frequency of 6OXY-specific IgM^high B cells prior to immunization correlated with vaccine efficacy in the 6OXY-KLH group. Immunization increased frequencies of 6OXY-specific B cell subsets D) IgM^high, E) swIg, and F) GC. Increased frequency of 6OXY-specific IgM^high B cells 14 days after the first immunization correlated to greater vaccine efficacy on oxycodone distribution to G) serum, H) brain, and I) oxycodone antinociception. Frequencies are the % of total lymphocytes in the bound fraction after positive enrichment of blood. The MPE% represents hind paw flicks. Data are mean±SEM. Data include three independent experiment with a total n=12 each group. ### p<0.001 compared to naïve; ** p<0.01 compared to KLH control; *** p<0.001 compared to KLH control.

Figure 6. CD4⁺ T cells-dependent B cell activation on vaccine efficacy

Selective depletion of CD4⁺ T cells prevented activation of 6OXY-specific B cells and subsequent 6OXY-KLH effects in C57Bl/6 mice. A) Depletion of CD4⁺ T cells, but not CD8⁺ T cells in peripheral lymph nodes and spleen. T cells are shown as log₁₀. B) The frequency of 6OXY-specific IgM^high and swIg B cells in enriched blood 14 days after the first immunization. C) Depletion of CD4⁺ T cells blunted vaccine efficacy against oxycodone distribution measured on day 35, 7 days after the last immunization. D) In C57Bl/6 mice, an increased number of 2W1S-specific CD4⁺ T cells in spleen biopsy prior to immunization correlated to subsequent greater efficacy against oxycodone in mice vaccinated with 6OXY-OVA^2W. Data are mean±SEM. Data consists of two independent experiments for a total of (A-C) n=6 and (D) n=9 each group. *** p<0.001 compared to control or as indicated by brackets.

Figure 7. Frequency of hapten-specific B cells prior to immunization in mice

6OXY-specific naïve B cells (% of total B cells in enriched sample) in pooled mesenteric and peripheral lymph nodes and spleen (n= 6), spleen biopsy (n= 24) or blood (n=24) from naïve BALB/c mice. The frequency of 6OXY-specific B cells in lymph nodes and spleens (LN+LP) was calculated from a previously published study (23). B cell analysis in spleen biopsy and blood was performed in three independent experiments (8 mice each day). Mouse: A) 6OXY-specific IgM^high B cells, B) 6OXY-specific ASC B cells, C) 6OXY-specific GC B cells, and D) 6OXY-specific swIg B cells.