Evaluation of Montanide™ ISA 71 VG adjuvant during profilin vaccination against experimental coccidiosis

Abstract

Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus Montanide™ ISA 70 VG (ISA 70) or Montanide™ ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone, and comparative RNA microarray hybridizations were performed to ascertain global transcriptome changes induced by profilin/ISA 70 vs. profilin alone and by profilin/ISA 71 vs. profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Further, canonical pathway analysis identified 10 pathways that were associated with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Therefore, ISA 71 was selected as a candidate adjuvant in conjunction with profilin vaccination for in vivo disease protection studies. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. Finally, the levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. None of these effects were seen in chickens injected with ISA 71 alone indicating that the adjuvant was not conferring non-specific immune stimulation. These results suggest that profilin plus ISA 71 augments protective immunity against selective Eimeria species in chickens.

Figure 1. Comparison of the numbers of differentially altered transcript levels in spleen lymphocytes when comparing chickens immunized with profilin plus ISA 70 vs. profilin alone, profilin plus ISA 71 vs. profilin alone, or profilin plus ISA 70 vs. profilin plus ISA 71.

(A) The numbers of up-regulated and down-regulated transcripts. (B) The number of identical transcripts.

Figure 2. IPA canonic pathway analysis of differential transcript levels in chickens immunized with profilin plus ISA 70 vs. profilin alone and profilin plus ISA 71 vs. profilin alone.

Illustrated are the pathways that were significantly associated with genes encoding the modulated transcripts in the comparisons of profilin plus ISA 70 vs. profilin alone (2 pathways) and profilin plus ISA 71 vs. profilin alone (10 pathways). The left ordinate and bars illustrates the statistical significance of each pathway expressed as the -log₁₀ (P value) calculated using the right-tailed Fisher exact test. The right ordinate and line illustrate the ratio of the number of genes from the dataset that map to the indicated pathway divided by the total number of genes within that particular pathway.

Figure 3. Effect of vaccination with profilin plus ISA 71 on body weight gain following infection with E. acervulina, E. tenella, or E. maxima.

Chickens were subcutaneously vaccinated with PBS, profilin alone, ISA 71 alone, or profilin plus ISA 71 at 7 and 14 days post-hatch. At 7 days post-secondary vaccination, the chickens were uninfected or infected with 1.0×10⁴ sporulated oocysts of E. acervulina (E.A), E. tenella (E.T), or E. maxima (E.M). Body weight gains were measured between 0 and 10 days post-infection. Each bar represents the mean ± S.D. value (n = 8). Within each graph, bars with different letters are significantly different according to the Duncan’s multiple range test (P<0.05).

Figure 4. Effect of vaccination with profilin plus ISA 71 on fecal oocyst shedding following infection with E. acervulina, E. tenella, or E. maxima.

Chickens were vaccinated and infected as described in Figure 3. Fecal oocyst numbers were measured between 6 and 10 days post-infection. Each bar represents the mean ± S.D. value (n = 8). Within each graph, bars with different letters are significantly different according to the Duncan’s multiple range test (P<0.05).

Figure 5. Effect of vaccination with profilin plus ISA 71 on serum anti-profilin antibody levels following infection with E. acervulina, E. tenella, or E. maxima.

Chickens were vaccinated and infected as described in Figure 3. Serum anti-profilin antibody levels were measured by ELISA at 3 days post-infection. Each bar represents the mean ± S.D. value (n = 5). Within each graph, bars with different letters are significantly different according to the Duncan’s multiple range test (P<0.05).

Figure 6. Effect of vaccination with profilin plus ISA 71 on intestinal IEL cytokine transcript levels following infection with E. acervulina, E. tenella, or E. maxima.

Chickens were vaccinated and infected as described in Figure 3. Intestinal IEL transcripts for IFN-γ (A), IL-2 (B), IL-10 (C), and IL-17A (D) were measured by quantitative RT-PCR at 3 days post-infection and normalized to GAPDH transcript levels. Each bar represents the mean ± S.D. value (n = 3). Within each graph, bars with different letters are significantly different according to the Duncan’s multiple range test (P<0.05). NS, not significant.