Characterizing TLR4 agonist EmT4™ as an anti-Mycobacterium tuberculosis vaccine adjuvant

Abstract

Tuberculosis (TB) is again the deadliest infectious disease globally, and more efficacious vaccines are needed to reduce this mortality. Successful subunit TB vaccines need antigens and adjuvants that are immunogenic, inexpensive, and accessible. Here we evaluated the potential of synthetically produced Monophosphoryl lipid A (SyMLP), a TLR4-agonist, formulated in an oil-in-water emulsion (EmT4™) in combination with selected fusion proteins, to drive an effective vaccine-mediated immunogenic response in C57BL/6 mice against Mycobacterium tuberculosis (M.tb) HN878 and H37Rv challenge. We first observed that EmT4™ enhances activation of C57BL/6 bone-marrow derived macrophages and dendritic cells measured by CD40, CD86, and MHCII expression by flow cytometry. EmT4™ did not induce safety signals in a scaled tolerability study. In immunogenicity studies, mice immunized 3 times 3 weeks apart with ID93 antigen + EmT4™ produced a significantly higher magnitude of circulating proinflammatory cytokines and ID93-specific immunoglobulin G (IgG) antibodies pre- and post-challenge with M.tb than saline control animals. Ex vivo ID93 restimulated splenocytes and lung cells elicited significant polyfunctional CD4+ T-helper 1 responses. Importantly, ID93 + EmT4™ immunizations significantly reduced bacterial burden in C57BL/6 mice 4 weeks post-challenge. Interestingly, EmT4™ paired with a next generation protein fusion ID91 also afforded prophylactic protection against M.tb HN878 challenge in both young (6 to 8 wk) and aged (20 mo) immunocompromised Beige mice. These protection and immunogenicity findings suggest that synthetically derived EmT4™ adjuvant is not only suitable to help backfill the preclinical TB vaccine candidate pipeline but is also suitable for the needs of the global community.

Keywords: Mycobacterium tuberculosis; TB; adjuvant; mouse model; vaccine.

Figure 1.

Comparison of natural core structure of LPS with SyMPL. Extensive similarity between MPL derived from naturally occurring LPS (left) and the synthetic TLR4 ligand (SyMLP, right) can be seen (box). To produce 3D-MPL^® (center), LPS from S. Minnesota R595 (left) is treated with acid and base hydrolyzing some of the ester bonds and leading to a mixture of multiple acyl-species of MPL, the active form of which is shown above. Note. L-Ara4N = 4-amino-4-deoxy-L-arabinose; PtdEtn = phosphatidylethanolamine; KDO = 3-deoxy-D-manno-octulosonic acid.

Figure 2.

EmT4™ adjuvant activates innate immune cells and enhances antigen presentation. Activation of bone marrow derived dendritic cells (BMDC) and macrophages (BMDM) with EmT4™ adjuvant. BMDC (A–C) and BMDM (D–F) from naïve C57BL/6 mice were stimulated with increasing concentrations (0.1–100 ng/mL) of EmT4™ (closed diamonds), LPS (open circle) or PBS (open square), and surface stained for activation markers after 48 h. CD11c+ BMDCs and CD11b+ BMDMs were evaluated for the percentage of cells co-expressing CD40 (A, D), CD86 (B, E) and MHCII (C, F). Data shown are mean ± SEM from n = 5 technical replicates per treatment, representative of 2 experiments. Asterisks indicate statistical significance of EMT4™ treatment compared to the PBS group at each dose, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using two-way ANOVA with Šídák's multiple comparisons test.

Figure 3.

Maximum tolerated dose study. (A) C57BL/6 mice were given a single immunization with 5 µg (pink squares), 10 µg (teal triangles), or 15 µg (purple diamonds) of EMT4™ and weighed through day 12 along with untreated cohorts (black circles). (B) In a separate study cohorts were given 4 consecutive immunizations with 5 µg of EMT4™ (purple squares), or 5 µg of a commercial TLR4 agonist (blue hexagons) and weighed through day 12 along with untreated cohorts (black circles). Dosing intervals are denoted by open upside down triangles along the X-axis. Treated cohorts were individually compared to untreated using a 2-way ANOVA with Dunnetts multiple comparisons, significant differences are denoted where *P < 0.05. Data representative of one experiment with n = 4 animals per group.

Figure 4.

Overview of experimental design and immunizations. (A) Schematic of the ID93 fusion antigen comprising 4 M.tb proteins with the molecular weight of each component. (B) Cohort design of vaccine regimen per group. (C) Timeline of immunizations and aerosol M.tb HN878 and M.tb H37Rv challenge. Experimental samples were collected at 2 time points: D69 (4 wk post-third immunization) and D98 (4 wk post- M.tb challenge). Immunogenicity timepoints included n = 4 mice per group whereas protection endpoints included n = 7 per group. Experiment performed one time for each M.tb challenge.

Figure 5.

Vaccination with ID93+EmT4™ induces robust circulating cytokine responses post-immunization and is still detectable post-M.tb HN878 challenge. Systemic cytokine levels were measured in serum samples collected 4 wk after the third immunization and 4 wk after challenge with M.tb HN878. Concentration of (A) IL-2, (B) TNF-α, (C) IFN-γ, (D) IL-1β, (E) IL-6, (F) IL-5, (G) IL-12p70, (H) IL-10, (I) IL-17A/F, (J) IL-15, and (K) IL-33 pg/mL post-immunization (left) and post-challenge (right). Bars show mean ± SEM, dots represent individual mice, n = 3-4/group. Asterisks indicate statistical significance compared to the saline group, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using 2-way ANOVA with Tukey’s multiple comparisons test.

Figure 6.

Vaccination with ID93+EmT4™ induces strong circulating chemokine responses post-immunization and is detectable post-M.tb HN878 challenge. Circulating chemokine levels were measured in serum samples collected 4 wk after the third immunization and 4 wk after challenge with M.tb HN878. Concentration of (A) KC/GRO, (B) IP-10, (C) MCP-1, (D) MIP-1α, and (E) MIP-2 pg/mL post boost immunization (left) and post challenge (right). Bars show mean ± SEM, dots represent individual mice, n = 4/group. Asterisks indicate statistical significance compared to the saline group, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using two-way ANOVA with Tukey’s multiple comparisons test.

Figure 7.

Vaccination with ID93+EmT4™ drives ID93 antigen-specific humoral and cellular immune responses post-immunization and post-M.tb HN878 challenge. Serum samples were collected 4 wk after the third immunization and 4 wk after infection with M.tb HN878 and were evaluated for: ID93 antigen-specific (A) Total IgG, (B) IgG1, (C) and IgG2c responses. Log₁₀ endpoint titer (EPT) is shown (LOD = limit of detection of the assay). Bars show mean ± SEM, dots represent individual mice, n = 4/group. Asterisks indicate statistical significance compared to the saline group, where ****P < 0.0001 using 2-way ANOVA with Tukey’s multiple comparisons test. Splenocytes (4 wk after the third immunization) and cells from lung homogenates (4 wk after infection with M.tb HN878) were cultured and stimulated with ID93 ex vivo and evaluated for CD4⁺ T cell responses by intracellular cytokine staining flow cytometry. ID93-stimulated polyfunctional (expressing IFN-γ, IL-2, TNF, or a combination of these cytokines) CD4⁺ T_H1 T cells (D) in the spleen post-immunization, and (E) in the lung post M.tb HN878 challenge. Bars show mean ± SEM, n = 4/group. Asterisks indicate statistical significance compared to the saline group, where **P < 0.01 and ****P < 0.0001 using 1-way ANOVA with Tukey's multiple comparisons test.

Figure 8.

ID93+EmT4™ provides prophylactic pulmonary protection against 2 M.tb isolates at 4 wk post-challenge. Cohorts of mice receiving saline, BCG, EmT4™ or ID93+EmT4™ vaccination regimens were then challenged with a LDA of either M.tb HN878 (A-B) or M.tb H37Rv (C, D) 4 wk after the third immunization. Bacterial burden was assessed by colony forming units (CFU) in lung (A, C) and spleen (B, D) organ homogenates 4 wk after challenge. Bars show the mean ± SEM and dots represent individual mice (n = 5-7/group). Asterisks indicate statistical significance compared to the saline group, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using 1-way ANOVA with Dunnett’s multiple comparisons test.

Figure 9.

ID91+EmT4™ vaccination provides prophylactic protection against M.tb HN878 in both young and aged Beige mice. (A) The 4 M.tb proteins comprising the ID91 antigen, and immunizations received by each group. (B) Timeline of immunizations and aerosol M.tb HN878 challenge. Experimental samples were collected at D70 (4 wk post M.tb HN878 challenge). Bacterial burden in (C) lung and (D) spleen organ homogenates of young (6 to 8 wk) and aged (20 mo) Beige mice immunized with either saline (circles) or ID91+EmT4 (squares and grey bar) post M.tb HN878 challenge. Bars show mean ± SEM, dots represent individual mice, n = 6-7/group. Asterisks indicate statistical significance compared to the saline group, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using 1-way ANOVA with Dunnett’s multiple comparisons test. Data representative of 1 experiment.