Ecological investigations of silkworms have revealed that Eri silkworms (Samia cynthia ricini) possess useful morphological and ecological characteristics for virus-like particle (VLP) production, namely non-seasonal breeding, longer lengths, and heavier weights than Bombyx mori silkworms. Furthermore, when vector DNA from Bombyx mori nuclear polyhedrosis virus (BmNPV), which is unable to replicate in Sf9 cells from Eri silkworms, was replaced with the Autographa californica nuclear polyhedrosis virus (AcNPV) vector, three improved AcNPV influenza virus recombinants capable of replication in Sf9 cells were obtained. Although VLP antigens produced previously in silkworms were not evaluated individually, the present recombinant Fukushima (FkH5) and Anhui (AnH7) VLP antigens were detected in tissue fluids and fat bodies of Eri silkworms. Here, we aimed to determine the function of the AcNPV vector and P143 gene by expressing recombinants in Sf9 cells and eri silkworm pupae. The FkH5 recombinant produced high yields of haemagglutinin (HA)-positive VLPs, showing a mean HA titre of 1.2 million. Similarly, high production of H7 HA VLPs was observed in the fat bodies of eri silkworm pupae. Antigenic analysis and electron microscopy examination of Eri-silkworm-produced H5 HA VLPs showed characteristic antigenicity and morphology similar to those of the influenza virus. Although FkH5 recombinants possessing the AcNPV vector did not replicate in Bm-N cells, the introduction of the helicase p143 gene from BmNPV resulted in their production in Bm-N and Sf9 cells.
To produce monovalent and bivalent influenza vaccines composed of virus-like particles (VLPs) containing hemagglutinin (HA), we generated four recombinant Baculoviruses derived from Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa california nuclear polyhedrosis virus (AcNPV). Monovalent Fukushima (A/tufted duck/Fukushima/16/2011 [H5N1]) (FkH5) and Anhui (A/Anhui/1/2013 [H7N9]) (AnH7) VLP influenza vaccines were produced in silkworm pupae infected with FkH5-BmNPV or AnH7-BmNPV. To produce a bivalent FkH5 and AnH7 vaccine, the pupae were simultaneously inoculated with FkH5-BmNPV and AnH7-BmNPV. Then, interleukin (IL)-containing bivalent vaccines were produced by Eri silkworm pupae following triple infection with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. Fluorescent antibody tests in Sf9 cells triple-infected with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV showed coexpression of FkH5, AnH7, and IL-12 antigens, suggesting the presence of VLPs containing all three antigens. We then performed competitive hemagglutination inhibition (CHI) tests to calculate the VLP vaccine constituents. Inoculation with two recombinant viruses led to the production of bivalent vaccines containing very similar amounts of the H5 and H7 antigens, suggesting that our dual infection system can be used to produce bivalent VLP vaccines. Immunisation of mice with our developed monovalent and bivalent VLP vaccines induced the production of HI antibody, which protected against a sublethal dose of influenza virus. These IL-12-containing vaccines tended to display increased protection against hetero-subtype influenza viruses.
We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300–500 μg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.
To counter the spread of multiple Japanese encephalitis virus (JEV) variants harboured in alternative host species and highly neurotoxic variants with new antigenicity, such as genotype V (Muar), methods for developing more effective and low-cost vaccines against a variety of epidemic JEV strains are required. Here, we successfully synthesized large amounts of a Muar virus-like particle (MVLP) vaccine for JEV in silkworm pupae by using a Bombyx mori nuclear polyhedrosis virus recombinant consisting of JEV codon-optimized envelope (E) DNA. In particular, histopathological examination suggested that MVLP was efficiently synthesized in body fat tissues as well as epithelial cells. Quantitative analysis indicated that one silkworm pupa produced 724.8 µg of E protein in the MVLP vaccine. Electron microscopic examination of purified MVLP vaccine defined a typical MVLP morphological structure. Detailed MVLP antigen assessment by immune-electron microscopy revealed that the majority of MVLPs were covered with approximately 10 nm projections. Boosted immunization with MVLP antigens in mice and rabbits tended to show improved plaque inhibition potency against homologous Muar and heterologous Nakayama, but less potency to Beijing-1 strains. Notably, mixed immune rabbit antisera against Nakayama and Muar VLP antigens led to an increase in the low antibody reaction to Beijing-1. Additionally, a stopgap divalent JEV vaccine consisting of MVLP and Nakayama VLP and its immune mouse serum significantly increased plaque inhibition titre against Muar, Nakayama and Beijing-1 strains. These findings suggested that low-cost MVLP vaccines prepared in silkworm pupae are suitable for providing simultaneous protection of individuals in developing countries against various JEV strains.
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