Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens

Abstract

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Keywords: LPS; NDV; TLR4; TLR7; adjuvant; combination; resiquimod.

Figure 1

Hemagglutination inhibition antibody titers in SPF chicken following immunization and challenge. Treatment effect was analyzed at each time point by two-way ANOVA with Tukey’s test as post hoc test. The probability of an alpha error was set at 0.05. Different superscripts above bars (mean ± SE) indicate significant difference between groups (p < 0.05). dpi: days post-NDV immunization; dpc: days post-challenge. Group A: Control; group B: Commercial NDV vaccine; group C: inactivated NDV vaccine; group D: inactivated NDV vaccine with LPS; group E: inactivated NDV vaccine with R-848; group F: inactivated NDV vaccine with LPS (25 µg) + R-848 (25 µg) and group G: inactivated NDV vaccine with LPS (50 µg) + R-848 (50 µg).

Figure 2

NDV-specific antibody titers in SPF chicken following immunization and challenge. Treatment effect was analyzed at each time point by two-way ANOVA with Tukey’s test as the post hoc test. The probability of an alpha error was set at 0.05. Different superscripts above bars (mean ± SE) indicate significant difference between groups (p < 0.05). dpi: days post-NDV immunization. Group A: Control; group B: Commercial NDV vaccine; group C: inactivated NDV vaccine; group D: inactivated NDV vaccine with LPS; group E: inactivated NDV vaccine with R-848; group F: inactivated NDV vaccine with LPS (25 µg) + R-848 (25 µg) and group G: inactivated NDV vaccine with LPS (50 µg) + R-848 (50 µg).

Figure 3

Lymphocyte transformation specific to NDV antigen in PBMCs collected from SPF chicken following immunization. Treatment effect was analyzed at each time point by two-way ANOVA with Tukey’s test as post hoc test. The probability of an alpha error was set at 0.05. Different superscripts above bars (mean ± SE) indicate significant difference between groups (p < 0.05). Group A: control; group B: commercial NDV vaccine; group C: inactivated NDV vaccine; group D: inactivated NDV vaccine with LPS; group E: inactivated NDV vaccine with R-848; group F: inactivated NDV vaccine with LPS (25 µg) + R-848 (25 µg) and group G: inactivated NDV vaccine with LPS (50 µg) + R-848 (50 µg).

Figure 4

Percentage of CD4⁺ and CD8⁺ T cells in PBMCs collected from SPF chicken analyzed by flow cytometry. Treatment effect was analyzed at each time point by two-way ANOVA with Tukey’s test as post hoc test. The probability of an alpha error was set at 0.05. Different superscripts above bars (mean ± SE) indicate significant difference between groups (p < 0.05). dpi: days post-NDV immunization. Group A: control; group B: commercial NDV vaccine; group C: inactivated NDV vaccine; group D: inactivated NDV vaccine with LPS; group E: inactivated NDV vaccine with R-848; group F: inactivated NDV vaccine with LPS (25 µg) + R-848 (25 µg) and group G: inactivated NDV vaccine with LPS (50 µg) + R-848 (50 µg).

Figure 5

Survival analysis (Kaplan–Meyer analysis) of experimental SPF chicken following challenge with virulent Newcastle disease virus. The survival curve was statistically different (p < 0.0001) with a chi-square value of 73.13 and df 6. Group A: control; group B: commercial NDV vaccine; group C: inactivated NDV vaccine; group D: inactivated NDV vaccine with LPS; group E: inactivated NDV vaccine with R-848; group F: inactivated NDV vaccine with LPS (25 µg) + R-848 (25 µg) and group G: inactivated NDV vaccine with LPS (50 µg) + R-848 (50 µg).