Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance

Influenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication

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

Induction of immune responses following immunization with VSV-ΔP expressing H1 and H7 HA.

<p>BALB/c mice (5 mice/group) were intravenously injected with 1×10⁷ pfu of VSVs in 100 μl at days 0 and 21. At day 28, sera were harvested to determine for H1N1-specific and H7N9-specific IgG levels at a titer of 5,120. Values are averages of two independent experiments with error bars showing SD.</p

Decreased lethality in mice after intracranial injection with VSV-ΔP.

<p>ICR mice (5 mice/group) were lightly anesthetized with ether and then intracranially injected with either PBS or 1×10⁴ pfu of VSVs. (A) Body weight was measured daily and (B) survival was plotted using the Kaplan-Meier survival curve. Values are averages of five mice with error bars showing SD and are representative of two independent experiments. NS, not significant; *, p<0.5; **, p<0.05.</p

Construction of recombinant VSVs with P gene deletion.

<p>(A) The top schematic shows the VSV genome layout and the naturally occurring restriction sites used for cloning. To construct VSV-ΔP, the VSV genome was digested with <i>Eco</i>RV, religated in the absence of the P gene fragment and the mCherry gene was then inserted between the G and L genes. The mCherry gene was replaced by HN, H1 or H7 HA to generate VSV-ΔP-HN, VSV-ΔP-HA1 and VSV-ΔP-HA7, respectively. (B) Supporting cells (BHK-P) were constructed by transducing BHK-21 cells with lentivirus bearing the P gene, and the selected clone expressing the emerald fluorescent protein was examined by (C) bright field and (D) fluorescence imaging. (E) BHK-P cells were infected with VSV or VSV-ΔP, and supernatants were harvested for viral genome extraction and RT-PCR. RBZ, hepatitis virus delta ribozyme; T7, T7 RNA polymerase leader; T7 ter, T7 terminator; LTR, long terminal repeat; ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; SFFV, spleen focus-forming virus (promoter); WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔU3, U3 deletion.</p

P gene deletion attenuated replication of recombinant virus.

<p>(A) BHK-21 and BHK-P cells were infected with VSV-ΔP at an MOI of 1 and observed for cytopathic effects (CPE). Infected cells were then subjected to flow cytometry to quantify the percentage of mCherry-expressing cells. The pictures are representative of triplicate samples. (B) BHK-P cells were infected with VSV or VSV-ΔP at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing standard deviation (SD). (C) Viruses were serially diluted for plaque titration, and plaques were stained with neutral red for visualization. Representative images of VSV and VSV-ΔP were selected for plaque size comparison.</p

Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.

<p>Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.</p

Expression of foreign antigens on the viral surface.

<p>(A) BHK-P cells were infected with VSVs at an MOI of 0.1. Supernatants were harvested at the indicated time points and were assessed using a MUNANA-based assay, (B) HA assay and (C) HAI assay. Supernatant and cell lysates were subjected to Western blot analysis using a β-actin monoclonal antibody as the primary antibody. Values are averages of triplicate wells with error bars showing SD. (D) 1×10⁷ purified VSVs were lysed and subjected to Western blot analysis using an HA (H1N1) polyclonal antibody and serum from VSV-immunized mice as the primary antibody. (E) To study the incorporation of the H7 HA gene in VSVs, RNA were extracted from purified VSVs and subjected to RT-PCR using primers specific for the N/M fragment or H7 HA genes. (F) BHK-P cells were infected with VSVs at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing SD. A.U., arbitrary units.</p