Interferon (IFN)-sensitive and replication-incompetent influenza viruses are likely to be the alternatives to inactivated and attenuated virus vaccines. Some IFN-sensitive influenza vaccine candidates with modified non-structural protein 1 (NS1) are highly attenuated in IFN-competent hosts but induce robust antiviral immune responses. However, little research has been done on the manufacturability of these IFN-sensitive vaccine viruses.
Prof. LIU Wenjun’s group at the Institute of Microbiology, Chinese Academy of Sciences (IMCAS) has been working on the replication mechanism ofinfluenza virus for many years.
In the present study, they generated an IFN-sensitive and replication-limited recombinant A/WSN/33 (H1N1) virus expressing the NS1 R38AK41A mutant in RIG-I-knockout 293T cells. The package efficiency of the NS1 R38A/K41A virus in RIG-I-knockout 293T cells was much higher than that in 293T cells.
Moreover, the NS1 R38A/K41A virus almost lost its IFN antagonist activity and could no longer replicate in A549, MDCK, and Vero cells after 3-6 passages. This indicated that the replication of NS1 R38A/K41A virus is limited in conventional cells.
Therefore, they further established a stable Vero cell line expressing the wild-type (WT) NS1 of the WSN virus, based on the Tet-On 3G system. The NS1 R38A/K41A virus was able to steadily propagate in this IFN-deficient cell line for at least 20 passages. In a mouse model, the NS1 R38A/K41A virus could not survive in mice for long periods of time because of the robust innate immune response.
Additionally, this virus induced high-titer neutralizing antibodies against the WT WSN, A/Puerto Rico/8/1934 (PR8), or A/California/04/2009 (CA04) viruses and provided 100% protection against the WT WSN virus.
Collectively, the replication of the NS1 R38A/K41A virus was limited in IFN-competent cells and mice. They presented a promising IFN-deficient system, involving a RIG-I-knockout 293T cell line to package the IFN-sensitive vaccine virus and a stable Vero cell line expressing NS1 to propagate the IFN-sensitive vaccine virus. The IFN-deficient system is applicable for the manufacture of IFN-sensitive vaccine virus.
This study has been published online in Frontiers in Cellular and Infection Microbiology (https://www.frontiersin.org/articles/10.3389/fcimb.2018.00127/full?) with Associate Prof. SUN Lei and Prof. LIU Wenjun as corresponding authors, Dr. CHEN Can as the first author.
This work was supported by grants from the National Key R&D Program of China, the National Natural Science Foundation of China, and the Strategic Priority Research Program of Chinese Academy of Sciences.