Lyophilized Listeria monocytogenes vaccines for storage at non-ultra-low temperatures and cancer therapy 2249461

Abstract

Abstract Introduction Lyophilization (freeze-drying) is a common method of preparing bacteria for storage at higher temperatures with lyoprotectant and buffer selection greatly impacting viability. Listeria monocytogenes (LM) is a Gram-positive intracellular bacterium being investigated preclinically and clinically as an anticancer vaccine platform and is stored at ultra-low temperatures (ULT). ULT storage greatly limits LM therapy accessibility globally due to a lack of necessary storage equipment in many areas of the world. Methods Here, we investigated preparations of lyophilized (freeze-dried) LM for stability at cold-chain viable temperatures while maintaining the required infectivity for anticancer effect. Using a murine subcutaneous renal cell carcinoma model we further compared the antitumor efficacy of non-lyophilized versus lyophilized LM LLO-CD105A vaccine and the effects of lyophilization on immune recruitment to the tumor microenvironment. Results Through development of lyophilization media, we found that the choice of lyoprotectant and buffer dramatically impacts LM viability following lyophilization and during storage. Of those tested, the HEPES 2.5% sucrose medium (3mM HEPES 2.5% w/v sucrose) demonstrated the best viability while maintaining the infectivity and antitumor efficacy of LM LLO-CD105A vaccine in a subcutaneous renal cell carcinoma model. Lyophilized LM demonstrated near identical efficacy to non-lyophilized LM but resulted in higher myeloid-derived suppressor cell infiltration into the tumor microenvironment. Conclusion The methods described herein are easily adoptable in both laboratory and industry settings for the preparation of LM and stable storage at non-ultra-low temperatures. These methods address a critical need for translation of LM cancer therapies for global distribution and enable further development of novel LM therapeutic approaches. Funding Source Laura W. Bush Institute for Women’s Health, TTUHSC Office of Research and Innovation Topic Categories Technological Innovations in Immunology (TECH)