Haemophilus influenza type b. [1] Chemical conjugation to CRM197 renders poly-/ oligo-saccharides immunogenic. [2] Poorly immunogenic peptides were also conjugated to CRM197 and became immunogenic. [3] However, conjugate vaccines are one of the most expensive vaccine types as they require laborious manufacture of pure recombinant CRM197 and pure polysaccharide or peptide followed by chemical conjugation and by removal of conjugation chemicals. [2,4] It should be noted that conjugate vaccine formulations include adjuvants to further enhance immunogenicity.Soluble protein antigens are safe but poorly immunogenic and require formulation with adjuvants to boost immunogenicity. [5] Such subunit vaccines composed of soluble protein antigens were approved to prevent such as, for example, whooping cough. Only a few adjuvants including aluminium hydroxide, MF59, and AS01 have been approved for use in vaccine formulations and there are concerns of adverse reaction caused by adjuvants. [6] Another but still experimental approach is formulation of soluble antigens into particles, which further enhances immunogenicity due to facilitated uptake by antigen-presenting cells (APCs), which activate T cell immune responses essential for protection against intracellular pathogens such as viruses and some bacteria (e.g., Mycobacterium tuberculosis). [7] Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A costeffective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

Currently available vaccines fail to provide consistent protection against tuberculosis (TB). New, improved vaccines are urgently needed for controlling the disease. The mycobacterial antigen fusions H4 (Ag85B-TB10.4) and H28 (Ag85B-TB10.4-Rv2660c) have been shown to be very immunogenic and have been considered as potential candidates for TB vaccine development. However, soluble protein vaccines are often poorly immunogenic, but augmented immune responses can be induced when selected antigens are delivered in particulate form. This study investigated whether the mycobacterial antigen fusions H4 and H28 can induce protective immunity when assembled into particulate vaccines (polyester nanoparticle-H4, polyester nanoparticle-H28, H4 nanoparticles and H28 nanoparticles). The particulate mycobacterial vaccines were assembled inside an engineered endotoxin-free production strain of Escherichia coli at high yield. Vaccine nanoparticles were purified and induced long-lasting antigen-specific T cell responses and protective immunity in mice challenged by aerosol with virulent Mycobacterium tuberculosis. A significant reduction of M. tuberculosis CFU, up to 0.7-log10 protection, occurred in the lungs of mice immunized with particulate vaccines in comparison to placebo-vaccinated mice (p < 0.0001). Polyester nanoparticles displaying the mycobacterial antigen fusion H4 induced a similar level of protective immunity in the lung when compared to M. bovis bacillus Calmette-Guérin (BCG), the currently approved TB vaccine. The safe and immunogenic polyester nanoparticle-H4 vaccine is a promising subunit vaccine candidate, as it can be cost-effectively manufactured and efficiently induces protection against TB.