Testing the Antigenic Potential of Transmembrane Proteins To Develop a Thermostable Tuberculosis MOF-Liposomal Vaccine

Abstract

Tuberculosis is one of the deadliest infectious diseases and continues to be a major health risk in many parts of the world. Even today, the century-old Bacillus Calmette-Guerin (BCG) vaccine is the only formulation on the market and is ineffective for several sections of the global population responsible for transmission. In the search for antigens that can mount a robust immune response, we have reported the recombinant expression and purification of two novel membrane proteins, the Cation transporter protein V (CtpV) and the Mycobacterial copper transporter B (MctB) present on the membrane surface of Mycobacterium tuberculosis. CtpV was tested as an antigen against the plasma of tuberculosis patients and was found to have a unique immune response profile compared with more commonly studied tuberculosis (TB) antigens. CtpV and MctB were reconstituted into proteoliposomes─individually and in combination─to stabilize them in a lipid bilayer and create a nanoparticle vaccine platform. In vivo experiments demonstrated that when delivered with an adjuvant, these antigens generated a robust Th1-biased T-cell response in mice, with the combination of both antigens performing the best and generating a response comparable to BCG. Since tuberculosis vaccines often need to be shipped to areas with fluctuating power supply, we encapsulated the proteoliposomes and the adjuvant in ZIF-8 to create a shelf-stable formulation. Complementary in vivo studies were carried out to confirm that the ZIF-8 coating did not interfere with or compromise the immunogenicity of the antigens.

Keywords: biomimetic mineralization; intranasal delivery; membrane protein antigens; metal−organic frameworks; tuberculosis vaccine.

Figure 1.

Purification and characterization of proteoliposomes. Size exclusion chromatogram of (A) CtpV and (B) MctB, and corresponding analysis by (C) SDS-PAGE gel electrophoresis (theoretical masses: CtpV −82.1 kDa, MctB −34.5 kDa). (D) DLS of MctB, CtpV, and Combo proteoliposomes. (E) Gel electrophoresis of all three proteoliposomes. The incorporation efficiency of the proteins was determined using gel band densitometry.

Figure 2.

Luminex data showing CtpV, PPD, and ESAT6CFP10-specific IgG titers. Plasma IgG levels were measured using a customized Luminex assay over three dilutions, and the area under the curve (AUC) was calculated. Each column represents an individual patient. Titers for (A) CtpV, (B) purified protein derivative (PPD), and (C) ESAT-6 & CFP-10 were determined in latent (n = 18) and active TB (n = 19) plasma samples as well endemic negative controls (n = 8). Dashed lines represent the median values for latent TB (blue), active TB (red), endemic negative controls (green), and saline background (gray).

Figure 3.

In vivo study of individual and combination antigens. (A) Experimental timeline for the study. Cytokines (B) TNF-α, (C) IL-2, (D) IFN-γ, and (E) IL-17A were measured on days 21, 28, 35, and 42. Antibody titers of (F) total IgG, (G) IgG1, and (H) IgG2a were measured on day 28, and the IgG2a/IgG1 ratio was used to derive the (I) Th1/Th2 ratio. Statistical analysis was performed using student’s t test with a confidence interval of 95%.

Figure 4.

Flow cytometry analysis of (A, B) spleen, (C, D) lymph nodes, and (E, F) lungs of mice after being sacrificed on day 42. Statistical analysis was performed using student’s t test with a confidence interval of 95%.

Figure 5.

(A) Synthetic scheme for the encapsulation of the Combo inside ZIF-8 (C@Z). Characterization of C@Z using (B) PXRD and (C) SEM (scale bar = 1 μm).

Figure 6.

Comparison of C@Z’s performance in vivo with Combo and BCG. Cytokines (A) TNF-α, (B) IL-2, (C) IFN-γ, and (D) IL-17A were measured on days 21, 28, 35, and 42. Antibody titers of (E) total IgG, (F) IgG1, and (G) IgG2a were measured on day 28. Flow cytometry analysis of (H, K) spleen, (I, L) lymph nodes, and (J, M) lungs of mice after being sacrificed on day 42. Statistical analysis was performed using student’s t test with a confidence interval of 95%.

Scheme 1.

Utilization of Membrane Proteins as TB Antigens^a a Transmembrane copper transporter proteins found on the outer (MctB) and inner (CtpV) membranes were recombinantly expressed in Escherichia coli. The proteins were purified as detergent micelle complexes. CtpV micelles were tested as antigens against the plasma of TB-positive patients, and their immune response in the form of TB-specific IgG and IgM was analyzed using a Luminex assay. CtpV and MctB were reconstituted into proteoliposomes—individually and in combination (Combo)—whose lipid bilayers mimic the native environment of transmembrane proteins. Combo was encapsulated in ZIF-8 (Combo@Z) to create a shelf-stable formulation. The immune profiles of CtpV, MctB, Combo, and Combo@Z—along with saline and BCG as controls—were evaluated in vivo in a mouse model using ELISA and flow cytometry.