A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model.

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3' and 5' UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.

An unconventional BST-2 function: down-regulation of transient protein expression.

While viral inhibition by tethering of budding virions to host cell membranes has been focused upon as one of the main functions of BST-2/tetherin, BST-2 is thought to possess other functions as well. Overexpression of BST-2 was found here to down-regulate transient protein expression. Removal of the N- and C-terminal regions of BST-2, previously described to be involved in signal transduction, reduced the impact of BST-2. These results suggest that BST-2-mediated signaling may play a role in regulating the levels of transiently expressed proteins, highlighting a new function for BST-2 that may also have implications for viral inhibition.

Improved transient protein expression by pFluNS1 plasmid.

Influenza virus nonstructural protein-1 (NS1) is abundantly expressed in influenza virus infected cells. NS1 is well recognized for counteracting host antiviral activities and regulating host and viral protein expression. When used as a plasmid component in DNA transfection, NS1 was shown to significantly increase expression levels of a cotransfected gene of different plasmid. Our previous studies demonstrated that addition of an NS1 plasmid increased the expression levels of influenza virus secreted neuraminidase (sNA) gene in 293T cells. In this study, we improved the utilization of NS1 as an enhancer for transient protein expression by generating pFluNS1 plasmid to contain two expression cassettes; one encoding an NS1 gene and another encoding a gene of interest. pFluNS1 is expected to codeliver the NS1 gene into the same cells receiving the gene of interest. The plasmid is therefore designed to induce higher protein expression levels than a cotransfection of an NS1 plasmid and a plasmid containing a gene of interest. To test the efficiency of pFluNS1, influenza virus sNA and non-viral DsRed genes were cloned into pFluNS1. The expression of these genes from pFluNS1 was then compared to the expression from a cotransfection of an NS1 plasmid and an expression plasmid coding for sNA or DsRed. We found that gene expression from pFluNS1 reached equal or higher levels to those derived from the cotransfection. Because the expression from pFluNS1 needs only one plasmid, a lesser amount of transfection reagent was required. Thus, the use of pFluNS1 provides a transfection approach that reduces the cost of protein expression without compromising high levels of protein expression. Together, these data suggest that pFluNS1 can serve as a novel alternative for an efficient transient protein expression in mammalian cells.

Evaluation of in vitro cross-reactivity to avian H5N1 and pandemic H1N1 2009 influenza following prime boost regimens of seasonal influenza vaccination in healthy human subjects: a randomised trial.

INTRODUCTION: Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses. METHODS: In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose. RESULTS: Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated. CONCLUSION: Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01044095.

Enhanced expression of secretable influenza virus neuraminidase in suspension mammalian cells by influenza virus nonstructural protein 1.

Influenza neuraminidase (NA) is a major target for anti-influenza drugs. With an increasing number of viruses resistant to the anti-NA drug oseltamivir, functionally active recombinant NA is needed for screening novel anti-NA compounds. In this study, the secretable NA (sNA) head domain of influenza A/Vietnam/DT-036/05 (H5N1) virus was expressed successfully in human embryonic kidney (HEK-293T) cells and shown to be enzymatically active. The inclusion of a plasmid encoding nonstructural protein 1 (NS1) of influenza A/Puerto Rico/8/34 virus with the sNA plasmid in the cotransfection demonstrated an increase in H5N1 sNA expression by 7.4 fold. Subsequently, the sNA/NS1 cotransfection protocol in serum-free 293-F suspension cell culture was optimized to develop a rapid transient gene expression (TGE) system for expression of large amounts of H5N1 sNA. Under optimized conditions, NS1 enhanced H5N1 sNA expression by 4.2 fold. The resulting H5N1 sNA displayed comparable molecular weight, glycosylation, K(m) for MUNANA, and K(i) for oseltamivir carboxylate to those of H5N1 NA on the virus surface. Taken together, the NS1-enhancing sNA expression strategy presented in this study could be used for rapid high-level expression of enzymatically active H5N1 sNA in suspension mammalian cells. This strategy may be applied for expression of sNA of other strains of influenza virus as well as the other recombinant proteins.

Enhancement of reverse genetics-derived swine-origin H1N1 influenza virus seed vaccine growth by inclusion of indigenous polymerase PB1 protein.

The current pandemic of a novel swine-origin H1N1 influenza virus (S-OIV) highlighted the need to urgently develop vaccines that can be used in a rapid response against the pathogen. Reverse genetics has been employed as an alternative means for the generation of influenza seed vaccines. However, reassortant viruses containing 6 internal genes from A/PR/8/34 and the hemagglutinin (HA) and neuraminidase (NA) genes from S-OIV showed very slow growth characteristics, hampering the speed of vaccine production. Here, we showed that the reverse genetics-derived H1N1 could be rescued with sensible viral titer by replacing PB1 of A/PR/8/34 with that of S-OIV for plasmid transfection. The "5+3" reassortant viruses have shown higher growth rate after transfection compared to that of "6+2" reassortant. The difference between PB1 of S-OIV and that of A/PR/8/34 in terms of the enhancement of virus growth was possibly due to the augmentation of viral polymerase activity, but not the lack of functional PB1-F2. Furthermore, it was found that growth enhancement by PB1 was specific for reassortant harboring HA of S-OIV, suggesting that the slow growth property of S-OIV reassortant virus is possibly due to restrictions imposed by the HA gene.

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines.

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE(2) production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE(2) formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA(2), COX, and mPGES-1, in PGE(2) synthesis pathway, and that these enzymes contribute to the elevation of PGE(2) level when one COX isoform is absent.

A novel cycloheptapeptide exerts strong anticancer activity via stimulation of multiple apoptotic pathways in caspase-3 deficient cancer cells.

BACKGROUND: VR3848 is a novel cycloheptapeptide, isolated from a Euphorbiaceae plant, which can suppress proliferation of various tumor cells at nanomolar concentration. Due to its novelty and potency, the molecular process of tumor cell growth inhibition was investigated intensively. MATERIALS AND METHODS: MCF-7 cells, a caspase-3 deficient cancer cell line, were treated with VR3848. The genetic response was monitored using cDNA array analysis. RESULTS: Expression alterations of caspase, bcl-2 family members, death receptor, death adaptor, death ligands, stress response, cell cycle machinery, mitogen-activated protein kinases (MAPKs) and proto-oncogene were found which can be linked into three apoptotic pathways. The first was the death receptor-mediated pathway, which was confirmed by functional inhibition of caspase-8 and -10. The second pathway was via ER-stress apoptosis demonstrated by up-regulation of ER-stress genes and the release of caspase-12 into the cytoplasm. The third pathway involved mitochondrial membrane leakage which was elucidated by down-regulation of anti-apoptotic bcl-2 and an increased level of cytosolic apoptosis-inducing factor (AIF). Cell cycle arrest was observed which may have been a direct effect of VR3848 or a consequence of DNA strand breaks which in turn stimulated cell cycle arrest. CONCLUSION: VR3848 inhibited MCF-7 cancer cell growth through an activation of three related apoptotic pathways.

Potent in vitro antimalarial activity of metacycloprodigiosin isolated from Streptomyces spectabilis BCC 4785.

Bioassay-guided fractionation of the extract from the fermentation broth of Streptomyces spectabilis BCC 4785 led to the isolation of three principle antimalarial agents, metacycloprodigiosin, bafilomycin A(1), and spectinabilin. Metacycloprodigiosin exhibited potent in vitro activity against Plasmodium falciparum K1, with a 50% inhibitory concentration of 0.0050 +/- 0.0010 microg/ml, while its cytotoxicity was much weaker.