Advancing usability of an influenza hemagglutinin virus-like particle vaccine expressing a chimeric cytokine.

Vaccine efficacy of conventional influenza vaccines depend on the antigenic similarity between the selected vaccine strain and annual epidemic strain. Since the influenza virus evolves yearly, a vaccine which is independent from viral antigenic mutation is desired. We have developed chimeric cytokine (CC) and hemagglutinin (HA) incorporated virus-like particle (CCHA-VLP) as a universal influenza vaccine candidate. Using mouse models, it was shown that the vaccine provided broad-based protective activity against several types of human and avian influenza A viruses. In this report, nasal immunization and mixture form (CC- and HA-VLP) were tested to improve usability of this vaccine. Immunogenicity was evaluated by induction of IgG, IgA, and IFN-γ secreting cells. Protective activity was measured as mouse survival rate against lethal challenge with H1N1 and H5N1 viruses and against H3N2 virus by lung viral titer. Nasal immunization showed low immunogenicity and low protective efficacy, but the addition of a sesame oil adjuvant improved vaccine efficacy. Mixture form of CC- and HA-VLP showed comparable or higher vaccine efficacy when compared to the incorporated form, CCHA-VLP. These results contribute to improved usability, such as needle-less administration and easy HA subtypes alteration.

The potential of a universal influenza virus-like particle vaccine expressing a chimeric cytokine.

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.

Interleukin 12-containing influenza virus-like-particle vaccine elevate its protective activity against heterotypic influenza virus infection.

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.

Genetic and antigenic characterisation of influenza A(H3N2) viruses isolated in Yokohama during the 2016/17 and 2017/18 influenza seasons.

BACKGROUND: Influenza A(H3N2) virus rapidly evolves to evade human immune responses, resulting in changes in the antigenicity of haemagglutinin (HA). Therefore, continuous genetic and antigenic analyses of A(H3N2) virus are necessary to detect antigenic mutants as quickly as possible. AIM: We attempted to phylogenetically and antigenically capture the epidemic trend of A(H3N2) virus infection in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons. METHODS: We determined the HA sequences of A(H3N2) viruses detected in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons to identify amino acid substitutions and the loss or gain of potential N-glycosylation sites in HA, both of which potentially affect the antigenicity of HA. We also examined the antigenicity of isolates using ferret antisera obtained from experimentally infected ferrets. RESULTS: Influenza A(H3N2) viruses belonging to six clades (clades 3C.2A1, 3C.2A1a, 3C.2A1b, 3C.2A2, 3C.2A3 and 3C.2A4) were detected during the 2016/17 influenza season, whereas viruses belonging to two clades (clades 3C.2A1b and 3C.2A2) dominated during the 2017/18 influenza season. The isolates in clades 3C.2A1a and 3C.2A3 lost one N-linked glycosylation site in HA relative to other clades. Antigenic analysis revealed antigenic differences among clades, especially clade 3C.2A2 and 3C.2A4 viruses, which showed distinct antigenic differences from each other and from other clades in the antigenic map. CONCLUSION: Multiple clades, some of which differed antigenically from others, co-circulated in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.

Overexpression of a virus-like particle influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion.

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.

Development of a Japanese encephalitis virus genotype V virus-like particle vaccine in silkworms.

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.

Functional growth inhibition of influenza A and B viruses by liquid and powder components of leaves from the subtropical plant Melia azedarach L.

We evaluated the anti-influenza-virus effects of Melia components and discuss the utility of these components. The effects of leaf components of Melia azedarach L. on viruses were examined, and plaque inhibition tests were performed. The in vivo efficacy of M. azedarach L. was tested in a mouse model. Leaf components of Melia azedarach L. markedly inhibited the growth of various influenza viruses. In an initial screening, multiplication and haemagglutination (HA) activities of H1N1, H3N2, H5, and B influenza viruses were inactivated by the liquid extract of leaves of M. azedarach L. (MLE). Furthermore, plaque inhibition titres of H1N1, H3N2, and B influenza viruses treated with MLE ranged from 103.7 to 104.2. MLE possessed high plaque-inhibitory activity against pandemic avian H5N1, H7N9, and H9N2 vaccine candidate strains, with a plaque inhibition titre of more than 104.2. Notably, the buoyant density decreased from 1.175 to 1.137 g/cm3, and spikeless particles appeared. We identified four anti-influenza virus substances: pheophorbide b, pheophorbide a, pyropheophorbide a, and pheophytin a. Photomorphogenesis inside the envelope may lead to removal of HA and neuraminidase spikes from viruses. Thus, MLE could efficiently remove floating influenza virus in the air space without toxicity. Consistent with this finding, intranasal administration of MLE in mice significantly decreased the occurrence of pneumonia. Additionally, leaf powder of Melia (MLP) inactivated influenza viruses and viruses in the intestines of chickens. MLE and MLP may have applications as novel, safe biological disinfectants for use in humans and poultry.

Characterization of a Feline Influenza A(H7N2) Virus.

During December 2016-February 2017, influenza A viruses of the H7N2 subtype infected ≈500 cats in animal shelters in New York, NY, USA, indicating virus transmission among cats. A veterinarian who treated the animals also became infected with feline influenza A(H7N2) virus and experienced respiratory symptoms. To understand the pathogenicity and transmissibility of these feline H7N2 viruses in mammals, we characterized them in vitro and in vivo. Feline H7N2 subtype viruses replicated in the respiratory organs of mice, ferrets, and cats without causing severe lesions. Direct contact transmission of feline H7N2 subtype viruses was detected in ferrets and cats; in cats, exposed animals were also infected via respiratory droplet transmission. These results suggest that the feline H7N2 subtype viruses could spread among cats and also infect humans. Outbreaks of the feline H7N2 viruses could, therefore, pose a risk to public health.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

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.

Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: A possible oral vaccine.

In this study, we aimed to quantitatively compare the increased production of three H7 influenza virus-like particle (VLP) haemagglutinin (HA) with the use of a codon-optimized single HA gene in silkworm pupae. Recombinant baculovirus (Korea H7-BmNPV) could produce 0.40 million HA units per pupa, corresponding to 1832µg protein. The yield of the HA produced in larva was estimated to be approximately 0.31 million HA units per larva, and there were no significant differences between the three HA proteins. We could establish efficient recovery system of HA production in larvae and pupae with the use of three cycles sonication methods. Next, we compared yields of HA proteins from three different H7 and two H5 recombinant baculoviruses based on the amount of mRNA synthesized in BmN cells, suggesting that mRNA synthesis may be also a useful indicator for the production of HA. Based on HA titres from four recombinants, the yield of HA had a great influence on the codon-optimized effect and the characteristics of the viral HA gene. The recombinant containing codon optimized HA DNA of A/tufted duck/Fukushima/16/2011 (H5N1) did produce more than one million HA units, although another recombinant including of the wild H5N1 strain failed to show HA activity. Electron microscopy revealed the presence of large VLP and small HA particle in the heavy and light fractions. The purified VLPs reacted with the authentic anti-H7 antibodies and the antibodies prepared after immunization with the VLP H7 antigen. Also H5 and H7VLPs could produce HI antibody in chickens and mice with oral immunization. The antibodies elicited with oral immunization were confirmed in fluorescent antibody analysis and western blotting in Korea H5-BmNPV and H7HA-BmNPV recombinant infected BmN cells. Taken together, these findings provided important insights into future oral vaccine development.

The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.

We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 µg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases.

Molecular characterization and analysis of equine rotavirus circulating in Japan from 2003 to 2008.

Using a total of 2018 fecal samples collected between 2003 and 2008 from foals with diarrhea, the molecular epidemiology of group A equine rotaviruses circulating in Japan was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) typing and sequence analysis of the VP4 (P type) and VP7 (G type) genes. A total of 1149 samples showed positive reactions with RT-PCR, of which 462 samples (40.2%) were positive for G3 type, 502 samples (43.7%) were positive for G14 type, and 185 samples (16.1%) were positive for both G3 and G14 types. To examine P types, 59 G3 and 56 G14 positive samples were used. The majority of the samples (96.5%) were characterized as P[12] type. In a phylogenetic analysis, the VP4 gene of the P[12] type in Japan was found to be conserved for a long time. The VP7 sequences of the G3 type were found to be clustered in the same group as the HO-5 strain, which is a G3 strain that was isolated in 1982 in Japan. In contrast, the VP7 sequences of the G14 type, which were in circulation between 2003 and 2008, were clustered differently from those of the G14 type strains isolated in Japan in the late 1990 s. These results suggest that the VP7 gene of the G3 type has been conserved over 25 years, while the VP7 gene of the G14 type circulating between 2003 and 2008 appears to have re-emerged in or invaded Japan around 2000.

Oseltamivir-resistant influenza a viruses circulating in Japan.

Surveillance studies of the influenza viruses circulating in Europe and other countries in 2007 and 2008 have revealed rates of resistance to oseltamivir of up to 67% among H1N1 viruses. In the present study, we examined 202 clinical samples obtained from patients infected with H1N1 virus in Japan in 2007 and 2008 for oseltamivir resistance and found that three were oseltamivir resistant (1.5%). The 50% inhibitory concentrations (IC(50)s), as measured by a sialidase inhibition assay with these drug-resistant viruses, were >100-fold higher than those of the nonresistant viruses (median IC(50), 12.6 nmol/liter). The His274Tyr (strain N2 numbering) mutation of the neuraminidase protein, which is known to confer oseltamivir resistance, was detected in these three isolates. Phylogenetic analysis showed that one virus belonged to a lineage that is composed of drug-resistant viruses isolated in Europe and North America and that the other two viruses independently emerged in Japan. Continued surveillance studies are necessary to observe whether these viruses will persist.

Estimation models for the morbidity of the horses infected with equine influenza virus.

Estimation formulas for the morbidity of horses infected with equine influenza virus by linear regression, logistic regression and probit transformation were developed, using data from the outbreak at the Sha Tin Racing Track in Hong Kong in 1992. Using these formulas, we estimated the equine influenza virus morbidity rates at training centers belonging to the Japan Racing Association (JRA) in October 1997 and in October 1998. In 1998 JRA started a new vaccination program, and every horse must now be vaccinated twice per year. At that time, the vaccine included two US lineage virus strains, the A/equine/Kentucky/81 strain and the A/equine/La Plata/93 (LP93) strain, against equine type-2 influenza viruses; it did not include any EU lineage virus strains, such as A/equine/Suffolk/89 (SF89). Comparing the geometric mean (GM) values of hemagglutination inhibition (HI) titers between the LP93 strain and the SF89 strain in 1997 and in 1998, they both rose significantly at every age (p<0.05) by Wilcoxon test. Calculations by the simulation models show the morbidity rates for LP93 diminished from 0.439 (linear), 0.423 (logistic) and 0.431 (probit) to 0.276 (linear), 0.265 (logistic) and 0.271 (probit), respectively. On the other hand, the estimated morbidity rates for SF89 diminished only slightly from 0.954 (linear), 0.932 (logistic) and 0.944 (probit) to 0.946 (linear), 0.914 (logistic) and 0.927 (probit), respectively. Our simulation models could estimate the effect of the vaccine on each of the equine virus strains represented by the morbidity of infected horses. Thus, they are useful for vaccine evaluation.

Phylogenetic analysis of bovine viral diarrhea viruses using five different genetic regions.

Phylogenetic analysis of the five different regions (5' non-coding region (5'NCR), N(pro), E2, NS3 and NS5B-3'NCR) of 48 Japanese and reported bovine viral diarrhea virus (BVDV) genomes was performed. Japanese BVDVs were segregated into BVDV1 subdivided into six subgroups and BVDV2. One isolate, So CP/75, isolated in 1975 and previously proposed as subgroup 1e according to its 5'NCR sequence, was quite unique and formed an independent lineage in the tree of any region. Another isolate, 190CP, obtained from an experimental mucosal disease case was classified as subgroup 1e, defined by Becher et al. in the 5'NCR, N(pro) and E2 regions, whereas it was classified as subgroup 1a in the NS5B-3'NCR region. The genomic sequences of the American isolates ILLC and ILLNC obtained from the GenBank database were assigned into subgroup 1b in the 5'NCR, N(pro), E2 and NS5B-3'NCR regions, whereas they were assigned into subgroup 1a in the NS3 region, suggesting that recombination between the virus strains classified into different subgroups had occurred in an animal. These findings suggest that phylogenetic analysis of several genetic regions is useful for the further characterization of field BVDV isolates.