Attenuated Influenza Virions Expressing the SARS-CoV-2 Receptor-Binding Domain Induce Neutralizing Antibodies in Mice

Abstract

An effective vaccine is essential for controlling the spread of the SARS-CoV-2 virus. Here, we describe an influenza virus-based vaccine for SARS-CoV-2. We incorporated a membrane-anchored form of the SARS-CoV-2 spike receptor binding domain (RBD) in place of the neuraminidase (NA) coding sequence in an influenza virus also possessing a mutation that reduces the affinity of hemagglutinin for its sialic acid receptor. The resulting ΔNA(RBD)-Flu virus can be generated by reverse genetics and grown to high titers in cell culture. A single-dose intranasal inoculation of mice with ΔNA(RBD)-Flu elicits serum neutralizing antibody titers against SAR-CoV-2 comparable to those observed in humans following natural infection (~1:200). Furthermore, ΔNA(RBD)-Flu itself causes no apparent disease in mice. It might be possible to produce a vaccine similar to ΔNA(RBD)-Flu at scale by leveraging existing platforms for the production of influenza vaccines.

Keywords: RBD; SARS-CoV-2; influenza; intranasal; live attenuated vaccine; spike.

Figure 1

Design and characterization of a neuraminidase-deficient influenza virus containing receptor binding domain (RBD) of SARS-CoV-2 spike protein. (A) Schematic diagram of construct encoding SARS-CoV-2 Spike RBD with a eukaryotic signal peptide and the transmembrane region and cytoplasmic tail from murine B7.1 [29] in the neuraminidase (NA) segment of influenza virus. Sequence for RBD is flanked at the 5′ and 3′ ends by the noncoding and coding terminal packaging regions of the NA viral RNA (vRNA) segment from A/WSN/33. Start (ATG) and stop codons (TAA) are shown to indicate the region that is expressed in cells. Note that the schematic is drawn in the orientation of the positive-sense mRNA, not the negative sense vRNA. (B) Agarose gel of amplified NA and hemagglutinin (HA) segments from ΔNA(RBD)-Flu. For comparison, HA and NA segments from virus containing a wildtype NA segment are also shown. The expected sizes of bands are: wildtype NA = 1409 bp, ΔNA(RBD) = 1125 bp, and HA = 1765 bp. (C) The effect of introducing G379W and R453G into H3_Y98F HA on viral titers. TCID50/mL is shown for reverse genetics for viruses with no NA segment, or the indicated NA segment. Values shown represent titers from a single reverse genetics experiment. Limit of detection is indicated with a horizontal dashed line. Samples where no cytopathic effect was observed are indicated with “n.d.” (none detected).

Figure 2

Infection with ΔNA(RBD)-Flu leads to expression of RBD on the surface of 293T cells. Histograms, normalized to mode, of flow cytometry measurements showing RBD expression on the cell surface for: (A) Cells transfected with the mammalian expression plasmids HDM-SARS-Spike-delta21 or HDM_Spike_RBD_B7-1 and stained with ACE2 or (B) stained with CR3022 antibody, (C) Cells infected with ΔNA(RBD)-Flu or ΔNA(GFP)-Flu and stained with ACE2 or (D) CR3022 antibody. Data shown are from a single population of stained cells.

Figure 3

The ΔNA(GFP)-Flu virus induces neutralizing antibodies against influenza and SARS-CoV-2. (A) Schematic of timeline for intranasal infection and blood collection from mice. (B) Weight of individual mice was measured for the first seven days postintranasal infection to monitor disease severity for any signs of disease. Values shown are percent weight change from initial weight prior to infection on Day 0. Points are average and error bars are standard deviation of each group. Titers of neutralizing antibodies against (C) SARS-CoV-2 Spike-pseudotyped lentiviral particles at 14 and 21 days postinfection, and (D) influenza virus with the H3 HA from A/Aichi/2/1968 at 21 days postinfection. Y-axis values show reciprocal dilution of the fifty-percent inhibitory concentration (IC50). Blue circles denote titer from individual mice in each group, horizontal line indicates median titer for the group. Samples were run in duplicate. The dashed line indicates the limit of detection, points at that limit (white squares) are the lower bound. There are four mice in each group.