The Usefulness of an Influenza Virus-Like Particle (VLP) Vaccine Produced in Silkworm Pupae and Virosomes and Liposomes Prepared by Chemical Means: From Virosome to VLP and the Future of Vaccines

Abstract

In our quest for an ideal influenza vaccine in 1989, we successfully prepared a muramyl dipeptide (MDP)-liposome-containing influenza virus hemagglutinin (HA) and neuraminidase (NA) virosome vaccine. This MDP-virosome vaccine showed enhanced HA immunogenicity in mice and elicited superior antibody production in humans, and a high HI titer was retained for up to 2 years. Generation of a cellular immune response was also confirmed by the production of cytotoxic T lymphocytes in mice. In fact, these killer T cells reduced the titer of the H3N2, H1N1 and B virus in the lungs of infected mice. Based on the finding that this MDP-virosome vaccine could elicit both humoral and cellular immune responses (i.e., high antibody activity and killer T cells), which is a requirement for an ideal vaccine, we examined the possibility of preparing the VLP vaccine in silkworms. Here, we show that significant amounts of influenza VLPs could be successfully produced in silkworm pupae by using a chimeric (multiple hybrid) HA DNA. In fact, the HA titers in homogenates from the infected pupae reached a mean value of 0.8 million HA units (HAU), equivalent to approximately 2,000 μg of HA protein per pupa, which is more than 50-fold higher than that produced in embryonated chicken eggs. Analysis of the VLPs in the purified HA fractions showed that they were attractive structures ranging from 30 to 300 nm in diameter. The spikes on the surface of the VLPs were approximately 14 nm long, densely packed, and were very similar to those on actual influenza virus particles. Subsequent important evidence was symbolized in the following genome constellation. In the present study, we showed, for the first time, that VLPs could be synthesized in silkworms by injection of a single synthetic chimeric HA DNA. These VLPs reflected the characteristic structure of the influenza virosome and elicited immune responses that were much higher than those elicited by the MDP-liposome prepared in our previous study. Therefore, the use of chemical immunomodulators such as MDP should be considered in the future for the development of VLP vaccines.