Advax4 delta inulin combination adjuvant together with ECMX, a fusion construct of four protective mTB antigens, induces a potent Th1 immune response and protects mice against Mycobacterium tuberculosis infection

Abstract

Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b⁺) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb - associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.

Keywords: ECMX; TB; adjuvant; advax; delta inulin; tuberculosis; vaccine.

Figure 1.

Upper part of the figure: representative schematic time line of vaccinations. (A) Mice were immunized three times with 30 days intervals. Blood samples were collected 30 days after each vaccination. Two days after the first vaccination, mice were euthanized to collect right quadriceps muscle and draining lymph nodes. Thirty days after the last vaccination, mice were infected with Mtb. Forty-five days after Mtb challenge, mice were euthanized to collect lung and spleen for analysis. Lower part of the figure: representative histopathological images of quadriceps muscle tissues of immunized mice. BALB/c mice were immunized in the quadriceps muscle with the different formulations and euthanized after two days for morphological analysis. Saline group (B), recombinant protein alone (C), Advax4 alone (D), Advax4 plus recombinant protein (E). In (E) is possible to observe a leukocyte increase consistent with chemotaxis induced by the vaccine formulation rather than an inflammatory process. The results shown are representative of two independent experiments (N = 6, *p<0.05). H&E staining, x100 magnification. Arrows indicate increase of mononuclear cells.

Figure 2.

Specific rCMX cells present in the draining lymph nodes 2 days after injection. BALB/c mice inoculated with Saline, rCMX, Advax4, or Advax4 + CMX were euthanized after 2 days, and the draining lymph nodes were collected. Cells were stained with gp38, CD31, F4/80, CD11C. (A) Percentage of blood endothelial cells (BEC). (B) Percentage of lymphatic endothelial cells (LEC). (C) Percentage of activated macrophages (F4/80⁺/CD11b). The results shown are representative of two independent experiments (N = 6, *p < 0.05).

Figure 3.

Humoral Immune Response to CMX. Animals were bled 30 days after the first, second and third vaccinations. Antibody levels were measured after sera separation. (A) IgG1 levels induced by CpG-DNA + CMX, Advax4 + CMX and controls. (B) IgG2a levels induced by CpG-DNA + CMX, Advax4 + CMX and controls. (C) IgG1 levels induced by Advax4 + ECMX. (D) IgG2a levels induced by Advax4 + ECMX. (E) IgG2a/IgG1 ratio of all vaccine formulations. All vaccines induced high antibody levels since 30 days after the first vaccination. The results shown are representative of two independent experiments (N = 6; *, # p < 0.05). * Statistical difference between the analyzed group and the same group after the first vaccination. # Statistical difference with the saline group.

Figure 4.

Cellular immune response against rCMX 45 days after challenge. Thirty days after the third immunization, BALB/c mice were challenged with Mtb (10⁵ CFU per mice) and then euthanized after 45 days to evaluate the cellular response. (A) Anti-rCMX or anti-rECMX CD4⁺IFN-γ⁺ lung cells 45 days after challenge. (B) Anti-CMX or anti-ECMX CD4⁺IFN-γ⁺ splenocytes 45 days after challenge. The results shown are representative of two different experiments (N = 3; *p < 0.05). * Statistical difference between the analyzed group and the BCG group. # Statistical difference with the saline group.

Figure 5.

Representative histopathological images of lungs, 45 days post Mtb challenge (A) Saline, (B) infection control, (C) BCG, (D) Advax4, (E) Advax4 + CMX, (F) Advax4 + ECMX. H&E staining, x100 magnification. Inflammatory injury reduction is observed 45 days after challenge in the Advax4 + ECMX group.

Figure 6.

Lung bacillary load of mice infected with Mtb. After 45 days of infection, the immunized mice were euthanized and the superior left lobe of lung mice collected for Mtb colony quantification in 7H11 media (N = 3, *p < 0.05).