Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality

Abstract

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Keywords: Toll-like receptor 4 signalling; adenoviral vector-based vaccine; follicular T helper cell; germinal centre; humoral immunity.

Figure 1

Adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA) can induce potent antibody responses and elevate Toll‐like receptor 4 (TLR4) expression on CD11c⁺ cells. (a) C57BL/6 mice (n = 6 to n = 8) injected with 1 × 10⁷ pfu/mouse of Ad5‐PA, 5 μg PA with or without 100 μg alum and phosphate‐buffered saline,(PBS), respectively. At 14 days post immunization, PA‐specific antibody titre was determined by ELISA. (b) Mice injected with 5 μg PA, 1 × 10⁷ pfu/mouse Ad5, 1 × 10⁷ pfu/mouse Ad5‐PA, 1 × 10⁷ pfu/mouse Ad5 plus 5 μg PA, and PBS. Histograms show expression of TLR4 on CD11c⁺ DCs sorted from different groups of immunized mice at 24 hr post immunization, represented as mean fluorescence intensity (MFI). Data shown are representative of three replicate experiments. Error bars represent mean ± SEM. Analysis of variance with Bonferroni post hoc tests for pairwise comparison of individual columns were used. *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Anti‐PA antibody production and cytokine responses to adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA) are mediated by Toll‐like receptor 4 (TLR4) signalling. (a) At 14 day post immunization, protective antigen (PA) ‐specific antibody titre determined by ELISA. (b, c) Samples were isolated 24 hr post immunization, cytokines interleukin 6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) analysed. Data shown are representative of three replicate experiments. Error bars represent mean ± SEM. Unpaired two‐tailed Student's t‐test was used: *P < 0·05, **P < 0·01.

Figure 3

Toll‐like receptor 4 (TLR4) deficiency impairs B‐cell activation and antigen‐presenting cell function during adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA) immunization. (a) Representative flow cytometry plots exhibiting gating scheme for B cells (CD3⁻ CD19⁺) and myeloid dendritic cells (mDCs) (CD11c CD11b⁺ MHCII ⁺). Spleen cells were prepared as described. Following light‐scatter gating, dead cells and debris were excluded from total splenocytes using a dump channel; all indicated cell types were determined using the isotype control. (b, c) Expression of activation marker CD69 and maturation marker CD86. (d) Expression of CD11b on mDCs from mice immunized with Ad5‐PA, measured by flow cytometry and represented as mean fluorescence intensity (MFI). (e) DCs immunized with Ad5‐PA from wild‐type (WT) and TLR4 knockout (TLR4‐KO) co‐cultured with naive CD4⁺ T cells. T‐cell proliferation was determined by measuring BrdU incorporation. Data pooled from three independent experiments (total four to six mice per group). Error bars represent mean ± SEM. Unpaired two‐tailed Student's t‐test was used. *P < 0·05, **P < 0·01.

Figure 4

Toll‐like receptor 4 (TLR4) signalling is required for germinal centre GC response to adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA). (a) At 14 days after immunization, spleens from wild‐type (WT) and TLR4 knockout (TLR4‐KO) mice harvested, and frozen sections labelled with anti‐GL7 (blue) and anti‐IgD (red) to delimit GCs and follicles, respectively. Images acquired with a 10× lens. Boxed area indicates image to the right. Each group shows proportion of the follicular cell area occupied by GCs. Individual points denote individual spleen sections. (b) Total number of GC B cells (defined as B220⁺ CD4⁻ IgD⁻ GL7⁺) and (c) Total number of Bcl6⁺ T follicular helper Tfh (B220⁻ CD4⁺ CXCR5⁺ PD‐1⁺ Bcl6⁺) cells measured by flow cytometry 14 days after immunization. (d) Expression of ICOS on Tfh cells and (e) expression of Fas on GC B cells, represented as mean fluorescence intensity (MFI). Data pooled from three independent experiments (total five or six mice per group). Error bars represent mean ± SEM. Unpaired, two‐tailed Student's t‐test was used: *P < 0·05, **P < 0·01.

Figure 5

Long‐lived plasma cells (LLPC) in bone marrow after adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA) immunization positively regulated by Toll‐like receptor 4 (TLR4) signalling. (a) Identification of LLPCs (defined as CD138^hi CD43⁺ B220⁻) in bone marrow by flow cytometry at day 90 after immunization with Ad5‐PA. (b) ELISpot assay of PA‐specific IgG‐secreting cells at day 90 after immunization without further culture. Data pooled from three independent experiments (total five or six mice per group). Error bars represent mean ± SEM. Unpaired two‐tailed Student's t‐test was used: **P < 0·01, ***P < 0·001.

Figure 6

Elevated expression of Toll‐like receptor 4 (TLR4) is mediated by hexon and ablated by anti‐HMGB1 antibody. (a) C57BL/6 mice (n = 6) injected with 1 × 10⁷ pfu/mouse adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA), 5 μg PA plus 1 × 10⁷ pfu/mouse Ad5, or 10 μg MPLA, co‐administered with either 200 μg alum or AddaVax (1 : 1, v/v). PA‐specific IgG antibody measured 14 days after immunization. (b) Total number of germinal centre (GC) B cells per 10⁵ live splenocytes measured by flow cytometry 14 days after immunization with different formulations as described in Fig. 1(b). (c) Different groups of mice immunized with PA (5 μg), Ad5 (1 × 10⁷ pfu), and Ad5‐PA (1 × 10⁷ pfu). At 12 hr after immunization, mice were injected with control IgG (50 μg/mouse) and anti‐HMGB1 (50 μg/mouse). At 24 hr after immunization, spleens were harvested and single‐cell suspensions were prepared. Expression of TLR4 on CD11c⁺ cells was evaluated by flow cytometry and represented as mean fluorescence intensity (MFI). (d) Expression of TLR4 on CD11c cells 24 hr after immunization with hexon (10 μg/mouse), fibre (15 μg/mouse), Ad5 (10⁷ pfu/mouse), and Ad5‐PA (10⁷ pfu/mouse). Data were pooled from three independent experiments (total 5–6 mice per group). Error bars represent mean ± SEM. Analysis of variance with Bonferroni post tests for pairwise comparison of individual columns were used: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 7

Involvement of Toll‐like receptor 4 (TLR4) in multiple elements in adenovirus vector (AdV) ‐based vaccine. Adenovirus serotype 5‐based anthrax vaccine (Ad5‐PA), particularly its hexon component, up‐regulates surface TLR4 on dendritic cells (DCs), dependent on HMGB1, inducing germinal centre (GC) reactions and antibody responses. Activated myeloid DCs (mDCs) participate in innate responses to AdV‐based vaccine; activated B cells can further migrate to follicles to form the GC, together with CD4⁺ helper T cells. Co‐stimulatory factor ICOS, transcription factor Bcl6, and apoptosis‐related marker Fas all participate in development of the GC response to enhance antibody response.