Self-emulsifying adjuvant systems containing alpha-tocopherol for subunit vaccines

Abstract

Recombinant DNA technology has revolutionized vaccine antigen production yielding highly purified synthetic antigens that offer significant advantages such as higher yield, lower production costs and improved safety. However, their higher purity resulted in reduced immunogenicity compared to conventional vaccines, like whole-inactivated or live-attenuated, due to absence of inherent immunomodulatory components such as diverse pathogen-associated molecular patterns (PAMPs). Vaccine adjuvants are components that enhance the immune response to antigens with low immunogenicity. These adjuvants represent a wide range of materials from synthetic molecules to heterogenous extract of natural products and include particulates such as emulsions, nanoparticles and liposomes. Among the particulate adjuvants, emulsion adjuvants have been used since early 1930s to improve immune responses to vaccine antigens. Emulsion adjuvants used in clinic such as MF59 (used in Fluad®) and AS03 used (in Pandemrix®) have helped in reducing the antigen dose as well as the number of immunizations required to achieve the required immune response. These attributes make emulsion adjuvants favorable for use, especially with influenza antigens. Most of the emulsion adjuvants currently available in clinic or in development use high shear and energy consuming processes such as high-pressure homogenization and microfluidization. Such complex processes, requiring expensive equipment and strict manufacturing requirements, make it a limitation for production in developing countries leading to unavailability in these less-privileged regions of the world. The 2009 H1N1 pandemic was an example where not enough doses of vaccine and the adjuvant were available to dose the entire population. Thus, there certainly exists a need to make the adjuvant production simple and less-expensive to aide in efficient storage, distribution and availability in all parts of the world. Like MF59 and AS03, several other emulsion adjuvants have squalene oil as main component along with different surfactants to stabilize the oil droplets. AS03 also has α-tocopherol added as an immunomodulator. α-Tocopherol has been used as an immune supplement for several decades now and has been an active component of veterinary vaccine adjuvants. AS03 has shown better immune response profile compared to squalene-only emulsion adjuvants in preclinical (using Hepatitis B antigen in BALB/c mice) as well as clinical studies (using H7N9 antigen and comparison with MF59) suggesting role of α-tocopherol in providing additional immunostimulation. More recently, it was shown that AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. They showed that the adjuvant effect elicited by AS03 may be due to the metabolic changes induced by AS03 via stress kinase receptor IRE1-α in the monocytic cells. Despite these evidences, use of α-tocopherol has only been in AS03 hitherto. This work might serve as an established route for discovery of more potent novel adjuvants containing α-tocopherol. Developing simpler emulsion adjuvants containing α-tocopherol may allow to understand its role even better at a molecular and cellular level. In this project, we use self-emulsification, as an alternative simple and low energy process to microfluidization, to formulate α-tocopherol containing emulsion adjuvant using components of AS03. We first demonstrate that this new self-emulsified adjuvant system (SE-AS) shows immune response similar to AS03 when administered with inactivated Quadrivalent Influenza Vaccine (QIV) in female BALB/c mice. We then used a soluble recombinant antigen, CMV wild-type pentamer to demonstrate that SE-AS shows significantly higher humoral response compared to SEA160 (a squalene oil only emulsion) in C57BL/6 mice. This confirmed that emulsion adjuvants containing α-tocopherol improved the immune response to a soluble recombinant antigen compared to emulsion adjuvants without α-tocopherol. Adjuvanted vaccines are distributed as a two or three vial presentation which heavily relies on cold-chain for storage and release. Cold-chain distribution not only gets expensive but also makes it difficult to distribute vaccines in developing countries. As mentioned previously, there are constant efforts to improve storage and distribution of vaccines in less-privileged countries and creating a single vial vaccine with help eliminating the cold-chain making it less expensive to distribute vaccines in these countries. In this project, we used lyophilization to create a single vial SE-AS adjuvanted CMV pentamer vaccine for reconstitution. We demonstrated that a single vial freeze-dried vaccine can be created and showed that it generates comparable immune responses to the bed-side mixed liquid vaccine.