A Dual TLR Agonist Adjuvant Enhances the Immunogenicity and Protective Efficacy of the Tuberculosis Vaccine Antigen ID93

With over eight million cases of tuberculosis each year there is a pressing need for the development of new vaccines against Mycobacterium tuberculosis. Subunit vaccines consisting of recombinant proteins are an attractive vaccine approach due to their inherent safety compared to attenuated live vaccines and the uniformity of manufacture. Addition of properly formulated TLR agonist-containing adjuvants to recombinant protein vaccines enhances the antigen-specific CD4+ T cell response characterized by IFN-γ and TNF, both of which are critical for the control of TB. We have developed a clinical stage vaccine candidate consisting of a recombinant fusion protein ID93 adjuvanted with the TLR4 agonist GLA-SE. Here we examine whether ID93+GLA-SE can be improved by the addition of a second TLR agonist. Addition of CpG containing DNA to ID93+GLA-SE enhanced the magnitude of the multi-functional TH1 response against ID93 characterized by co-production of IFN-γ, TNF, and IL-2. Addition of CpG also improved the protective efficacy of ID93+GLA-SE. Finally we demonstrate that this adjuvant synergy between GLA and CpG is independent of TRIF signaling, whereas TRIF is necessary for the adjuvant activity of GLA-SE in the absence of CpG

Prophylactic efficacy against Mycobacterium tuberculosis using ID93 and lipid-based adjuvant formulations in the mouse model.

An estimated 10 million people developed tuberculosis (TB) disease in 2019 which underscores the need for a vaccine that prevents disease and reduces transmission. The aim of our current studies is to characterize and test a prophylactic tuberculosis vaccine comprised of ID93, a polyprotein fusion antigen, and a liposomal formulation [including a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant, GLA) and QS-21] in a preclinical mouse model of TB disease. Comparisons of the ID93+GLA-LSQ vaccines are also made to the highly characterized ID93+GLA-SE oil-in-water emulsion adjuvant, which are also included these studies. The recent success of vaccine candidate M72 combined with adjuvant AS01E (GlaxoSmithKline Biologicals) in reducing progression to active disease is promising and has renewed excitement for experimental vaccines currently in the TB vaccine pipeline. The AS01E adjuvant contains monophosphoryl lipid A (MPL) and QS-21 (a saponin) in a liposomal formulation. While AS01E has demonstrated potent adjuvant activity as a component of both approved and experimental vaccines, developing alternatives to this adjuvant system will become important to fill the high demand envisioned for future vaccine needs. Furthermore, replacement sources of potent adjuvants will help to supply the demand of a TB vaccine [almost one-quarter of the world's population are estimated to have latent Mycobacterium tuberculosis (Mtb) according to the WHO 2019 global TB report], addressing (a) cost of goods, (b) supply of goods, and (c) improved efficacy of subunit vaccines against Mtb. We show that both ID93+GLA-SE (containing an emulsion adjuvant) and ID93+GLA-LSQ (containing a liposomal adjuvant) induce ID93-specific TH1 cellular immunity including CD4+CD44+ T cells expressing IFNγ, TNF, and IL-2 (using flow cytometry and intracellular cytokine staining) and vaccine-specific IgG2 antibody responses (using an ELISA). In addition, both ID93+GLA-SE and ID93+GLA-LSQ effectively decrease the bacterial load within the lungs of mice infected with Mtb. Formulations based on this liposomal adjuvant formulation may provide an alternative to AS01 adjuvant systems

GLA and CpG combine to enhance the protective efficacy of ID93.

<p>Mice were immunized and challenged with a low dose of aerosolized <i>M.tb.</i> (A) four or (B) twelve weeks later. <i>M.tb.</i> burdens in the lungs were determined four weeks after infection. Statistically significant differences between vaccinated groups were determined by ANOVA using the Bonferroni correction for multiple comparisons. Data are representative of three experiments with similar results with seven animals per group.</p

GLA and CpG combine to enhance T_H1 responses to ID93.

<p>Mice were immunized with ID93 adjuvanted with GLA, CpG, or GLA+CpG. (A and B) One week after the final immunization ID93-specific CD4⁺ T cells were identified by staining with I-A^b tetramers presenting dominant epitopes from Rv2608 and Rv3619. Cells are gated as singlet, CD4⁺ CD44⁺. Splenic ID93-specific T_H1 CD4⁺ T cells from immunized mice were identified by cytokine production following <i>ex-vivo</i> restimulation with ID93 and analyzed for (C) total cytokine response or (D) poly-functional responses. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083884#pone.0083884.s001" target="_blank">Figure S1</a> for representative cytokine staining. Data are representative of four experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p

Enhanced T_H1 responses to GLA and CpG are independent of TRIF signaling.

<p>(A) C57BL/6 and TRIF^−/− splenocytes were stimulated <i>ex vivo</i> with GLA, CpG or both and analyzed for IL-6, IL-12p40, and TNF production by macrophages. (B and C) C57BL/6 and TRIF^−/− mice were immunized with ID93 adjuvanted with GLA, CpG or GLA+CpG. Splenic ID93-specific T_H1 CD⁺4 T cells from immunized mice were identified by cytokine production following ex-vivo restimulation with ID93. Data are representative of two experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p