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

Lyophilization is a common method of preparing bacteria for storage with lyoprotectant and buffer 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 limits LM therapy accessibility due to a lack of necessary storage equipment in many areas of the world. Here, we show lyophilization of LM maintains viability and efficacy of LM anticancer vaccines in a murine subcutaneous renal cell carcinoma model. 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 (3 mM HEPES 2.5 % w/v sucrose) demonstrated the best viability while maintaining the infectivity and antitumor efficacy of LM in a subcutaneous renal cell carcinoma model. The methods described herein are easily adoptable for the preparation of LM and stable storage at non-ultra-low temperatures.