Tuberculosis (TB) is a chronic infectious disease caused by respiratory infection by Mycobacterium tuberculosis (Mtb). It is the second single fatal infectious disease after new coronavirus pneumonia. Subunit vaccines are safe and highly immunogenic, and can provide specific immune protection against bacterial infections. Therefore, the development of safe and effective Mtb subunit vaccines is one of the important contents of TB research.

Liposome nanoparticles(LNP) are a type of vaccine adjuvant or delivery system with one or more concentric lipid bilayer structures. Due to their good biocompatibility and high bioavailability, they have been used in the prevention and treatment of important diseases such as COVID-19 and cancer. This study intends to build an LNP subunit vaccine that encapsulates the antigen protein EsxV and the mucosal adjuvant molecule c-di-AMP (EsxV:c-di-AMP:LNP,EsxV:C:L).

Figure 1 On the front page of the article

, weigh and add 20mg of cholesterol to 20mL of absolute ethanol, fully dissolve it in a water bath at 50 degrees Celsius, and then add 120mg of soy lecithin to fully dissolve it; the above mixed solution was put in a round-bottom flask at 50-60rpm and 50 degrees Celsius to remove the ethanol until a colorless and transparent lipid film was formed. Subsequently, 60ML phosphate buffer solution (PBS) at pH 7.0 was added, and 0.0048g of DSPE-PEG2000 was added. The round-bottom flask was rotated under a water bath of 52 degrees Celsius and normal pressure to hydrate and dissolve the lipid membrane. The water bath of 50 degrees Celsius was continued for 2 hours to formmulti-chamber liposomes. Sonicate in an ice water bath for 10 minutes, and the conditions were set as follows: sonication for 5 seconds, intermittent for 5 seconds, and power for 200W. After the ultrasound, single-chamber liposomes were obtained in a water bath at 50 degrees Celsius for 1 hour. Add 500μg EsxV antigen and 167μg c-di-AMP to the single-chamber liposome, and sonicate again under the same conditions to obtain EsxV:C:L. At the same time, blank LNP without EsxV and c-di-AMP was prepared according to the above conditions and stored at 4 degrees Celsius for later use.

Figure 2 LNP transmission electron microscope observation

Figure 3 LNP particle size distribution

Figure 4 LNP PDI value

Figure 5 LNP Zeta potential

The results showed that the blank LNP was basically spherical and uniform in size (Figure 2A); the shape of the LNP wrapped with EsxV and the adjuvant c-di-AMP did not change significantly (Figure 2B), and was consistent with the particle size and PDI test results. The above suggests that EsxV:C:L LNP and blank LNP are stable in shape and uniformly distributed, and can be used for later immunological characteristics research.

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