Immunization Strategies Producing a Humoral IgG Immune Response against Devil Facial Tumor Disease in the Majority of Tasmanian Devils Destined for Wild Release

Abstract

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government's Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials (N = 19 and N = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol^®) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.

Keywords: Tasmanian devil facial tumour disease; adjuvant; humoral immunity/antibody response; vaccination; wild immunology.

Figure 1

Flow cytometry histograms for three individual devils showing antibodies for MHC-I^-ve devil facial tumor disease cells in their pre-immune (gray) and post-immune (blue) serum samples. The fluorescence intensity is in log scale and shown on the x-axis, the cell count is on the y-axis. (A–C) demonstrate the range of responses found across the cohorts.

Figure 2

Serum anti-devil facial tumor disease IgG antibody levels (MFIR) for Narawntapu National Park devils showing effect of (A) protocol, (B) age, and (C) sex. The MFIR for each devil at each time point (2 weeks after primary course, on the day of the booster and 2 weeks after the booster) has been plotted on each graph. Protocol A = 4 immunizations at 4-week intervals, B = 4 immunizations at 4- or 6-week intervals, C = 3 immunizations at 4-week intervals, D = 2 immunizations at 2- or 4-week intervals. The p values for (A–C) were obtained with a four-way ANOVA analysis. See Table 3 for detailed ANOVA results. MFIR, median fluorescence intensity ratio.

Figure 3

Narawntapu National Park (NNP) devils’ serum anti-devil facial tumor disease IgG antibody levels (MFIR). (A) Responses of devils that had all four immunizations in their primary course, i.e., protocol A or B, Table 2. Only those devils for which sera samples were available at all time points are included. (B) Responses of all devils for each of three time points: end of primary immunization course, day of booster (4 months later) and 2 weeks post booster. Statistical analysis was performed with repeated-measures one-way ANOVA and only significant p values are shown on the graphs. See Table 5 for statistical details. Pre-immune MFIR for each devil is equal to 1 and is, therefore, not shown. MFIR, median fluorescence intensity ratio.

Figure 4

Serum anti-devil facial tumor disease IgG antibody levels (MFIR) of Stony Head devils after their 1st and 2nd immunizations. Pre-immune MFIR for each devil is equal to 1 and is, therefore, not shown. MFIR, median fluorescence intensity ratio.

Figure 5

Serum anti-devil facial tumor disease (DFTD) IgG antibody levels [median fluorescence intensity ratio (MFIR)] of (A) Stony Head (SH) and Narawntapu National Park (NNP) devils 4 weeks post 1st immunization; (B) SH and NNP devils just at the end of their primary immunization courses.

Figure 6

Serum anti-devil facial tumor disease (DFTD) IgG antibody levels [median fluorescence intensity ratio (MFIR)] of (A) Narawntapu National Park (NNP) devils post release compared to their post booster response in September 2015; (B) Stony Head (SH) devils after their primary course compared to 5 months later; and (C) NNP and SH devils, 5 months after their primary immunization course, just prior to the booster administration.