Nature Biotechnology: Generation of a live attenuated influenza A vaccine by proteolysis targeting

Posted by Ellen Burns on September 2nd, 2022

Longlong Si's research group from the Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, published a research titled "Generation of a live attenuated influenza A vaccine by proteolysis targeting" in Nature Biotechnology. Using influenza virus as a model virus, the team established the technology of protein degradation targeting virus as an attenuated vaccine (Proteolysis-Targeting Chimeric virus vaccine, PROTAC vaccine), which provides new ideas for vaccine development.

Virus infection and transmission endanger human health and socio-economic development. Influenza is a respiratory infection caused by the influenza virus. Influenza viruses are antigenically variable, spread rapidly, and can cause seasonal epidemics each year. Vaccines are one of the most cost-effective means of preventing and controlling infectious diseases. In 2021, Science listed "development of next generation vaccines" as one of 125 cutting-edge scientific issues (www.science.org/content/resource/125-questions-exploration-and-discovery). Attenuated vaccines have become one of the important development directions due to their potential advantages in terms of immune effects, for example, attenuated influenza vaccines can be inoculated by intranasal spray that is simpler, economical, painless, and consistent with the natural route of infection; they can retain the natural structure of all or most antigens of the virus and induce a wider immune response, including humoral immunity, respiratory mucosal immunity, and cellular immunity; and they can provide cross-immune protection.

As a common life substance necessary for the structural composition and normal life activities of viruses, proteins provide an important entry point for the study of manipulating viruses and then using viruses. Based on protein regulation, virus attenuation strategies can be roughly summarized into two main aspects: one is to inhibit or block protein synthesis to reduce the "raw materials" required for progeny virus assembly, and the other is to accelerate protein degradation and timely remove the "raw materials" required for progeny virus assembly. In this study, the research team constructed the PROTAC virus to reduce the replication ability of the virus by manipulating the degradation of viral proteins and attenuated the wild-type virus into a vaccine.

The naturally occurring protein degradation machinery "ubiquitin-proteasome system" in host cells lays a key biological foundation for the design of PROTAC viral vaccines. In recent years, PROTAC protein targeted degradation technology based on the ubiquitin-proteasome system has been used to develop chemical small molecule-based protein degradants and researchers have designed a small molecule compound with two active ends, one active end can bind to target proteins that need to be degraded, while the other active end can bind to specific E3 ubiquitin ligases, thereby inducing ubiquitination of target proteins, which are then degraded by the proteasome.

In this study, the team expanded the biological mechanism by which the host cell protein degradation machinery selectively degrades target proteins to the design and construction of animal-viral vaccines. The team chose influenza virus as a model virus and used the naturally occurring protein degradation machinery in host cells to design elements that conditionally manipulate the stability and degradation of viral proteins, engineered viral genomes, so that the corresponding viral proteins were recognized and degraded by the ubiquitin-proteasome system in normal cells, resulting in weakened viral replication ability and becoming potential vaccines; while in vaccine preparation cells, viral protein degradation-inducing elements would be selectively removed and viral proteins could be retained, so PROTAC virus could replicate efficiently and be prepared in large numbers in vaccine preparation cells.

According to the above design principle, the team first constructed a PROTAC influenza virus vaccine, named M1-PTD. Investigation of the virus growth curve showed that M1-PTD could only replicate efficiently in PROTAC virus preparation cells and could be prepared, while the replication ability was significantly decreased and safe in normal cells. In addition, immunofluorescence assay showed that M1-PTD viral protein was degraded in normal cells; plaque assay showed that M1-PTD could form plaques only in PROTAC virus-prepared cells, but not in normal cells; cytopathic assay showed that M1-PTD did not cause significant lesions in normal cells. These results suggest that M1-PTD influenza virus has the potential to be a safe vaccine.

The team validated the working mechanism of the constructed PROTAC influenza virus. The results showed that the protein of M1-PTD influenza virus was degraded in normal cells and replication was weakened, while the inhibition of proteasome in host cells could restore the viral protein level and replication ability of M1-PTD, indicating that the protein degradation and replication weakening of PROTAC influenza virus were ubiquitin-proteasome pathway-dependent and in line with the design principle.

The team evaluated the safety of the constructed M1-PTD influenza virus using a mouse, ferret animal model. Animals were inoculated intranasally with M1-PTD virus or wild-type influenza virus, monitored for mortality and body weight, and tested for viral titers in nasal washes, trachea, and lungs. The results showed that compared with wild-type virus, M1-PTD had significantly reduced replication ability in animals and did not cause death or weight loss in mice, indicating that it was safe in animals.

The team evaluated the immune efficacy of the M1-PTD influenza vaccine in mouse and ferret animal models. The results showed that M1-PTD could induce a wide range of immune responses, including humoral, mucosal and cellular immune responses, and M1-PTD could provide good cross-immune protection.

Based on the concept of synthetic biology, this study expands the biological mechanism of cell protein degradation machinery to the design of living body-virus vaccines, provides new ideas for viral vaccine development, enriches the arsenal of human vaccine technology against viruses, and helps to promote the deep cross-fusion of basic biological research of cell protein degradation machinery and medical transformation of vaccine research and development. At the same time, the team proposes that while the study demonstrates the feasibility of the PROTAC virus vaccine concept in cellular and animal models, the potential application of PROTAC virus as a vaccine still requires a lot of optimization and exploration.