Abstract
BackgroundInfluenza A viruses are major human and animal pathogens with huge economic and societal impact from illness, hospitalizations, and deaths. Virus-like particles (VLPs) of influenza virus have been suggested as a vaccine candidate offering improved safety and efficacy. To develop this concept further, we established a flexible platform to efficiently generate different subtypes of mammalian-expressed influenza VLPs. Here we demonstrate that these mammalian VLPs strongly resemble the authentic viruses in structure, particle size and composition of host factors, and even glycosylation of viral antigens.Methodology/Principal FindingsIn this study, a mammalian VLP system was established by stable co-expression of four influenza structural proteins (HA, NA, M1, and M2) in a Vero cell line. By replacing the surface glycoproteins of HA and NA, we converted the H3N2-VLP subtype to H5N1-VLP. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were identified by LC/MS/MS. Protease protection assays demonstrated that specific cellular proteins that co-purified with influenza virions were integrated into mammalian VLPs. The glycosylation profiles of mammalian VLPs as revealed by deglycosylation assays were similar to that of progeny viruses produced from Vero cells. Vaccination of mice with 2.5 µg and above of H5N1-VLP elicited H5-specific IgG1 antibodies and resulted in full protection against lethal infection with homologous virus. These results provide compelling evidence that mammalian VLPs closely emulate the exterior of authentic virus particles not only in antigen presentation but also in biological properties and should provide promising vaccine candidates.Conclusions/SignificanceThis flexible mammalian influenza VLP system offers a superior alternative to the conventional reverse genetic vaccine platform without concerns over inadequate presentation of immune antigens or limitations imposed by the manipulation of real viruses.
Highlights
Influenza infection is a major threat to human health and results in significant morbidity and mortality worldwide
We have developed a flexible platform for the efficient production of mammalian influenza Virus-like particles (VLPs) by coexpressing four influenza virus proteins, HA, NA, matrix 1 (M1), and matrix 2 (M2) in Vero cells
Our results suggest that VLPs generated from this platform will have similar glycosylation profiles to the authentic viruses that result from infection in the same host species
Summary
Influenza infection is a major threat to human health and results in significant morbidity and mortality worldwide. The manufacture of the current licensed influenza vaccines, either in the form of a split subvirion (disrupted, highly purified virus) or a subunit vaccine (purified hemagglutinin, HA, and neuraminidase, NA), is absolutely dependent on fertilized chicken eggs as a production bioreactor This method has substantial limitations since the manufacturing capacity is restricted by the availability of eggs, which may be insufficient to meet the urgent requirements for vaccine during a pandemic [1,2]. These vaccines induce antibodies primarily to the viral HA and are efficacious in healthy adults, but display lower protective rates in high-risk groups (e.g., the elderly) and may be poorly immunogenic in young children. We demonstrate that these mammalian VLPs strongly resemble the authentic viruses in structure, particle size and composition of host factors, and even glycosylation of viral antigens
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