BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis

Abstract

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4⁺ and CD8⁺ T cells and CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells, and produced higher IFN-γ responses in CD4⁺, CD8⁺, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Keywords: Burkholderia pseudomallei; IFN-γ; T-cell responses; melioidosis; type 2 diabetes; vaccine.

Figure 1

BpOmpW-activated T cells and induced effector CD4⁺ and CD8⁺ T cells. (A) Schematic illustration of the experimental timeline of the non-insulin-resistant mouse study. (B) Percentages of parent cells expressing CD45RB. (C–F) Percentages of CD4 and CD8 T cells expressing CD25 and CD44 activation markers. (G–K) Percentages of different populations of CD4 and CD8 T cells defined by different levels of CD45RB and CD44, such as effector CD4 T cells CD45RB^lowCD44^high (G), naive CD4 T cells CD45RB^highCD44^low (H), effector CD45RB^highCD44^high CD8 T cells (I), effector CD8 T cells CD45RBlow CD44^high (J), and naive CD8 T cells CD45RB^highCD44^low (K). SAS, splenocytes from adjuvant-only mice exposed to BpOmpW (orange circles, control) (n = 11); OmpW-SAS, splenocytes from SAS-adjuvanted BpOmpW-immunized mice re-exposed to BpOmpW in vitro (purple circles, treatment) (n = 11); BpOmpW, homologue OmpW in Burkholderia pseudomallei. Asterisks denote statistically significant differences according to a two-tailed t-test. The levels of significance are represented as follows: ****p < 0.0001.

Figure 2

BpOmpW-stimulated CD8^hi and CD4^hi populations that are CD45RB^hiCD44^lo. (A, B) Splenocytes of CD8^hi (in red) and CD4^hi (in blue) populations from both SAS-adjuvanted BpOmpW-immunized mice re-exposed to BpOmpW (n = 11) (A) and from adjuvant-only mice exposed to BpOmpW (n = 11) (B). (C, D) CD4 (C) and CD8 (D) T-cell co-expressions of CD45RB and CD44 in BpOmpW-re-exposed splenocytes from SAS-adjuvanted BpOmpW-immunized mice. (E, F) CD4 (E) and CD8 (F) T-cell co-expressions of CD45RB and CD44 in BpOmpW-exposed splenocytes from SAS-adjuvanted saline-immunized mice. (G, H) Naive CD4 and CD8 T cells (CD45RB^hiCD44^lo) including CD45RB^veryhi populations as naive T cells. BpOmpW, homologue OmpW in Burkholderia pseudomallei; SAS, Sigma Adjuvant System. The levels of significance are represented as follows: ****p < 0.0001.

Figure 3

BpOmpW immunization stimulated IFN-γ responses in CD4⁺, CD8⁺, natural killer (NK), and natural killer T (NKT) cells and upregulated regulatory T cells (Tregs). (A, B) Percentages of CD4 (A) and CD8 (B) T cells expressing IL-2 cytokine. (C–E) Percentages of CD4 T cells expressing IFN-γ (C), IL-4 (D), and IL-17 (E). (F) Percentages of CD8 T cells expressing IFN-γ. (G) Percentages of double-negative (DN) cells (CD4⁻CD8⁻). (H) Percentages of NKT cells. (I–K) Percentages of NKT cells expressing IFN-γ (I), IL-17 (J), and IL-4 (K). (L) Percentages of NK cells expressing IFN-γ. (M) Percentages of Tregs. (N, O) Percentages of CD4 (N) and CD8 (O) T cells expressing TNF. SAS, splenocytes from adjuvant-only mice exposed to BpOmpW (orange circles, control) (n = 11); OmpW-SAS, splenocytes from SAS-adjuvanted BpOmpW-immunized mice re-exposed to BpOmpW in vitro (purple circles, treatment) (n = 11). Asterisks denote statistically significant differences according to a two-tailed t-test. The levels of significance are represented as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Insulin-resistant mice mimicked with minimal changes the immune response to BpOmpW (homologue OmpW in Burkholderia pseudomallei) produced by non-insulin-resistant mice. (A) Percentages of the CD45RB marker in total T cells. (B–E) Percentages of CD4 and CD8 T cells expressing CD25 and CD44 activation markers. (F, G) Percentages of CD4 (F) and CD8 (G) T cells expressing IL-2 cytokine. (H–J) Percentages of CD4 T cells expressing IFN-γ (H), IL-17 (I), and IL-4 (J). (K–M) Percentages of CD8 (K), NKT (L), and NK (M) cells expressing IFN-γ. (N, O) Percentages of NKT cells expressing IL4 (N) and IL17 (O). (P) Percentages of regulatory T cells (Tregs). (Q, R) Percentages of CD4 (Q) and CD8 (R) T cells expressing the tumor necrosis factor (TNF) cytokine. SAS, splenocytes from adjuvant-only mice exposed to BpOmpW (orange circles, control) (n = 11); OmpW-SAS, splenocytes from SAS-adjuvanted BpOmpW-immunized mice re-exposed to BpOmpW in vitro (purple circles, treatment) (n = 11). Asterisks denote statistically significant differences according to a two-tailed t-test. The levels of significance are represented as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

BpOmpW (homologue OmpW in Burkholderia pseudomallei) induced the production of IFN-γ in HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice. Immunization of HLA-DR and HLA-DQ transgenic mice highlights HLA class II determining immunodominant epitopes of BpOMpW. (A–C) Mice transgenic for HLA-DR4 (n = 6), (A), HLA-DR1 (n = 6) (B), and HLA-DQ8 (DQB1*0302) (n = 6) (C) were primed with 25 μg rBpOmpW and the draining lymph node cells assayed with IFN-γ ELISpot in response to the indicated peptide on day 10. Data are plotted as spot-forming cells (SFCs) per 10⁶ cells for individual mice. (D, E) Responses to peptide were defined as positive if SFCs are greater than the mean + 2 SD of the response in the absence of any antigen (shown as horizontal dotted line): n = 6 (D) and n = 5 (E) transgenic mice. The levels of significance are represented as follows: *p < 0.05.

Figure 6

BpOmpW (homologue OmpW in Burkholderia pseudomallei) induced T-cell proliferation in human peripheral blood mononuclear cells (PBMCs). A table showing a qualitative description (YES/NO) based on the differential significance of CD4⁺ and CD8⁺ T-cell proliferation and the overall IFN-γ responses of 23 donor PBMCs in response to BpOmpW or BpOmpW+SAS and SAS control is included. The table also shows the human leukocyte antigen (HLA) alleles of each donor. YES/NO denote significant differences (p < 0.05) according to two-way ANOVA.

Figure 7

BpOmpW-specific immunoglobulin G (IgG) responses in plasma from melioidosis survivors. Detection by ELISA of the BpOmpW-specific IgGs in plasma from different cohorts: NE, non-endemic (n = 3); HH, healthy householders (n = 15); D, healthy diabetics (n = 15); M, melioidosis diabetic survivors (n = 15). BpOmpW, homologue OmpW in Burkholderia pseudomallei Asterisks mark significant differences according to two-tailed Student’s t-test. The levels of significance are represented as follows: *p < 0.05; **p < 0.01.