Fig. 3.

Whole-cell poly-LDN(F) vaccines are immunogenic and induce a sustained anamnestic response. (A) Freshly-thawed aliquots of 10⁶–10⁷ cells, or PBS, were delivered without adjuvant, intra-peritoneally to female Swiss-Webster mice and facial vein bleeds were collected according to the immunization scheme depicted. Three immunizations were given at 0, 2 and 4 weeks (1°), and one secondary (2°) boost was given at week 19, to the number of mice indicated under each timeline. The mice given PBS immunizations at weeks 0–4 made no detectable response and were therefore used for 1° immunization at 19 weeks. (B–E) To measure immunogenicity of each cell type, antisera from immunized mice was run on flow cytometry against fixed cells of the same type to assess IgM and IgG binding to surface antigens. (B) Histograms representative of two to three experiments are shown for the pooled antisera at week 0 (solid gray), week 6 (thick gray line) and week 20 (thick black line) post-immunization for IgM (left) and IgG (right). (C) The memory response was assessed by comparing pooled week 19 sera (pre-2°; solid gray) with shifts from week 20 sera from 2° immunized mice (thick black line) and 1° immunized mice (which had previously received PBS as mock immunization; dashed black line), representative of 2-3 experiments. (D) The increase in antibody binding to homologous cell types, expressed as fold change in average MFI relative to week 0, was measured for antisera pooled from each group of mice over time, representative of 3 experiments. Yellow triangles, Lec8-immunized vs. Lec8 cells; blue triangles, L8-GT-immunized vs. L8-GT cells; red triangles; L8-GTFT-immunized vs. L8-GTFT cells; open circles, PBS-immunized vs. Lec8 cells (only shown through week 6); black dash, mean for each group. (E) Flow cytometry was also used to assess the range of magnitudes in IgM (left) and IgG (right) response for individual mice, where enough serum was available. Two-way ANOVA with repeated measures was performed on 3-4 mice from each of the L8, L8-GT and L8-GTFT which had complete data at weeks 0, 6, 20 and 40. Within each group, time points were compared with week 0 using Dunnett's multiple comparisons test.