A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes.

Seasonal influenza vaccines offer little protection against pandemic influenza virus strains. It is difficult to create effective prepandemic vaccines because it is uncertain which influenza virus subtype will cause the next pandemic. In this work, we developed a nucleoside-modified messenger RNA (mRNA)-lipid nanoparticle vaccine encoding hemagglutinin antigens from all 20 known influenza A virus subtypes and influenza B virus lineages. This multivalent vaccine elicited high levels of cross-reactive and subtype-specific antibodies in mice and ferrets that reacted to all 20 encoded antigens. Vaccination protected mice and ferrets challenged with matched and mismatched viral strains, and this protection was at least partially dependent on antibodies. Our studies indicate that mRNA vaccines can provide protection against antigenically variable viruses by simultaneously inducing antibodies against multiple antigens.

Expression- and genomic-level changes during passage of four baculoviruses derived from bacmids in permissive insect cell lines.

The baculovirus-based bacmid expression vector system has been widely used for protein production in basic research and biotechnological laboratories. Since the first construction of the Autographa californica multiple nucleopolyhedrovirus bacmid (AcBacmid), three more bacmids have been created from Bombyx mori nucleopolyhedrovirus (BmBacmid), Spodoptera exigua nucleopolyhedrovirus (SeBacmid) and Helicoverpa armigera nucleopolyhedrovirus (HaBacmid). Each of these bacmid-derived viruses replicates efficiently in a range of specific and permissive cell types. Here, we investigated the relative stability of each virus derived from the bacmid during passage in permissive cell lines through assessment of their expression level and genome structure changes. Using two different reporters, the expression levels of the viruses from the AcBacmid-Sf9, AcBacmid-Tn5, BmBacmid-BmN and SeBacmid-SeE1 bacmid-cell systems were significantly reduced after five passages of the viruses, whereas the reductions were not detected in the AcBacmid-Sf21 and HaBacmid-HzAM1 systems. Pulse field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis of viral DNA isolated from passaged viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 systems showed no major genomic changes. In contrast, the genomes from passaged viruses in the AcBacmid-Tn5 and AcBacmid-Sf9 systems displayed reduced genome size and various mutations at individual loci, including genotypes missing one at least or more viral RNA polymerase subunits and fp25k. These genotypic changes were correlated with reduced protein expression. RFLP analysis of viral DNA from passaged viruses in the BmBacmid-BmN and SeBacmid-SeE1 systems exhibited changes in genome size, including excision of particular EcoRI fragments containing the mini-F replicon. Collectively, our data suggest that the viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 bacmid-cell systems are better for large-scale protein expression in continuous culture. Further study is needed to investigate the mechanism(s) behind the protein expression reduction in these bacmid-derived virus/cell systems.

Identification of human vaccinees that possess antibodies targeting the egg-adapted hemagglutinin receptor binding site of an H1N1 influenza vaccine strain.

Human influenza viruses passaged in eggs often acquire mutations in the hemagglutinin (HA) receptor binding site (RBS). To determine if egg-adapted H1N1 vaccines commonly elicit antibodies targeting the egg-adapted RBS of HA, we completed hemagglutinin-inhibition assays with A/California/7/2009 HA and egg-adapted A/California/7/2009-X-179A HA using sera collected from 159 humans vaccinated with seasonal influenza vaccines during the 2015-16 season. We found that ∼5% of participants had ≥4-fold higher antibody titers to the egg-adapted viral strain compared to wild type viral strain. We used reverse-genetics to demonstrate that a single egg-adapted HA RBS mutation (Q226R) was responsible for this phenotype.

Improved pFastBac™ donor plasmid vectors for higher protein production using the Bac-to-Bac® baculovirus expression vector system.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based Bac-to-Bac® expression system consists of a bacmid and five pFastBac™ donor transfer vectors. It has been widely used for eukaryotic gene expression in insect cells to elucidate gene function in biotechnology laboratories. The pFastBac™ vectors contain a 50bp AcMNPV polyhedrin (polh) promoter and a 127bp SV40 polyadenylation (pA) signal for cloning a gene of interest into the bacmid, resulting in unsolved lower gene expression levels than the wild type (wt) AcMNPV in insect cells. Therefore, the purpose of this research is to understand why the Bac-to-Bac system produces lower gene expression levels. Here, we determined that bacmids transposed with pFastBac™ vectors produced 3-4 fold lower levels of certain proteins than the wt AcMNPV. We found that an 80bp cis element 147bp upstream of the 50bp polh promoter and a 134bp polh pA signal are required in pFastBac™ to achieve bacmid protein expression levels equivalent to wt AcMNPV in High Five insect cells. Therefore, researchers currently using pFastBac™ vectors for protein expression can transfer their genes of interest into the improved vectors in this report to elevate protein expression yields in insect cells to reduce protein production costs.

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE: Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

Genomic sequence of Heliothis virescens ascovirus 3g isolated from Spodoptera exigua.

Heliothis virescens ascovirus 3a (HvAV-3a), a member of the family Ascoviridae, has the highest diversity among ascovirus species that have been reported in Australia, Indonesia, China, and the United States. To understand the diversity and origin of this important ascovirus, the complete genome of the HvAV Indonesia strain (HvAV-3g), isolated from Spodoptera exigua, was determined to be 199,721 bp, with a G+C content of 45.9%. Therefore, HvAV-3g has the largest genome among the reported ascovirus genomes to date. There are 194 predicted open reading frames (ORFs) encoding proteins of 50 or more amino acid residues. In comparison to HvAV-3e reported from Australia, HvAV-3g has all the ORFs in HvAV-3e with 6 additional ORFs unique to HvAV-3g, including 1 peptidase C26 gene with the highest identity to Drosophila spp. and 2 gas vesicle protein U (GvpU) genes with identities to Bacillus megaterium. The five unique homologous regions (hrs) and 25 baculovirus repeat ORFs (bro) of HvAV-3g are highly variable.

Genome of Thysanoplusia orichalcea multiple nucleopolyhedrovirus lacks the superoxide dismutase gene.

Thysanoplusia orichalcea multiple nucleopolyhedrovirus (ThorMNPV) has high virulence to Trichoplusia ni and Pseudoplusia includens larvae, with a potential for biological control of insect pests. The genome of ThorMNPV was sequenced and found to be 132,978 bp, with a G+C content of 37.9%. There are 145 predicted open reading frames (ORFs), encoding proteins of 50 or more amino acid residues with minimal overlap. Of the 145 ORFs, 141 appeared to be homologous to those of Autographa californica MNPV (AcMNPV). In comparison to AcMNPV, 9 ORFs of AcMNPV were absent in ThorMNPV, including the superoxide dismutase (sod) gene.

Phylogenetic position and replication kinetics of Heliothis virescens ascovirus 3h (HvAV-3h) isolated from Spodoptera exigua.

Insect-specific ascoviruses with a circular genome are distributed in the USA, France, Australia and Indonesia. Here, we report the first ascovirus isolation from Spodoptera exigua in Hunan, China. DNA-DNA hybridization to published ascoviruses demonstrated that the new China ascovirus isolate is a variant of Heliothis virescens ascovirus 3a (HvAV-3a), thus named HvAV-3h. We investigated the phylogenetic position, cell infection, vesicle production and viral DNA replication kinetics of HvAV-3h, as well as its host-ranges. The major capsid protein (MCP) gene and the delta DNA polymerase (DNA po1) gene of HvAV-3h were sequenced and compared with the available ascovirus isolates for phylogenetic analysis. This shows a close relationship with HvAV-3g, originally isolated from Indonesia, HvAV-3e from Australia and HvAV-3c from United States. HvAV-3h infection induced vesicle production in the SeE1 cells derived from S. exigua and Sf9 cells derived from S. frugiperda, resulting in more vesicles generated in Sf9 than SeE1. Viral DNA replication kinetics of HvAV-3h also demonstrated a difference between the two cell lines tested. HvAV-3h could readily infect three important insect pests Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius) from two genera in different subfamilies with high mortalities.