A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice

Abstract

Avian influenza H7N9 viruses continue to pose a great threat to public health, which is evident by their high case-fatality rates. Although H7N9 was first isolated in humans in China in 2013, to date, there is no commercial vaccine available against this particular strain. Our previous studies developed a replication-defective influenza virus through mutation of the hemagglutinin (HA) cleavage site from a trypsin-sensitive to an elastase-sensitive motif. In this study, we report the development of a reassortant mutant influenza virus derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], which is the QVT virus. The HA gene of this virus possesses three mutations at the cleavage site, Lys-Gly-Arg were mutated to Gln-Thr-Val at amino acid (aa) positions 337, 338, and 339, respectively. We report this virus to rely on elastase in vitro, possess unaltered replication abilities when elastase was provided compared to the wild type virus in vitro, and to be non-virulent and replication-defective in mice. In addition, we report this virus to induce significant levels of antibodies and IFN-γ and IL-5 secreting cells, and to protect mice against a lethal challenge of the BC15 (H7N9) virus. This protection is demonstrated through the lack of body weight loss, 100% survival rate, and the prevention of BC15 (H7N9) viral replication as well as the reduction of proinflammatory cytokines induced in the mouse lung associated with the influenza disease. Therefore, these results provide strong evidence for the use of this reassortant mutant H7N9 virus as a replication-defective virus vaccine candidate against H7N9 viruses.

Keywords: elastase dependent virus; influenza A virus H7N9; replication-defective virus vaccine.

Figure 1

The schematic outline of the mutations introduced into the HA cleavage site of BC15 (H7N9). (a) Nucleotide sequences from HA positions 1075 to 1086 and (b) amino acid sequences from HA positions 337 to 340 of WT HA (wild type) and HA/QTV (mutant plasmids). Nucleotide sequences (a) and amino acids (b) in red correspond to the mutations that were introduced. Elastase corresponds to the human neutrophil elastase protease.

Figure 2

The generation and characterization of the reassortant mutant QTV virus in relation to its replication-dependency and kinetics. (a,b) The replication-dependency of reassortant wild type (rWT) and reassortant mutant QTV viruses. MDCK cells were infected at an m.o.i. of 0.001 in the presence of 1 μg/mL TPCK-trypsin, 0.5 μg/mL human neutrophil elastase, or in the absence of an exogenous protease. The supernatant and cells were harvested at 48 h.p.i., and underwent either plaque assay (a) or Western Blotting (b) to detect the presence of nucleoprotein (NP) and matrix (M1) proteins. (c) The replication curve of the rWT and QTV viruses on MDCK cells. The cells were infected with the respective virus at an m.o.i. of 0.001 with either 1 μg/mL TPCK-trypsin (T) or 0.5 μg/mL human neutrophil elastase (E). The reassortant mutant QTV virus was also tested in the presence of TPCK-trypsin. The supernatants were collected at specified time points until 72 h.p.i., and then titered by plaque assay on MDCK cells.

Figure 3

The body weight, survival rate, and lung viral titration of mice infected with the rWT virus and the reassortant mutant QTV virus. Male and female BALB/c mice (n = 12 per group, equal males and females) were intranasally infected with either MEM (control), the rWT virus, or the reassortant mutant QTV virus at a single dose of 1 × 10³ PFU. The mice were monitored daily, with any mouse reaching a humane intervention point to be humanely euthanized and their lung samples harvested. (a) The body weights. (b) The survival rate. (c) The viral titration of homogenized lung samples at 3 d.p.i. by plaque assay. The samples were analyzed in duplicate. Viral titres are expressed as PFU per gram (PFU/gr). The data is shown as the mean ± the standard deviation. The dots correspond to the values obtained from individual mouse samples.

Figure 4

The outline of mice vaccinated with the reassortant mutant QTV virus and challenged with the homologous BC15 (H7N9) virus. BALB/c mice (n = 12 or 14, equal males and females) were intranasally vaccinated on days 0 and 21 with 50 μL of MEM (control) or the QTV virus (1 × 10³ PFU). On day 30, four mice per group (two males and two females) were euthanized for splenocyte isolation. On day 31, the remaining mice were intranasally challenged with MEM (control) or a lethal dose of BC15 (H7N9) (WT) (1 × 10³ PFU). On day 34 (three days post-challenge), four mice per group (two males and two females) were euthanized for sampling. The trial concluded on day 45 (14 days post-challenge), where the remaining mice were euthanized and sampled. Sampling included serum and lung tissue.

Figure 5

Antibody responses mounted after QTV vaccination. BALB/c mice were vaccinated on days 0 and 21 with MEM (control) or the QTV virus at a dose of 1 × 10³ PFU. Serum was collected on days 0, 21, and 30 to be titered using an ELISA to detect influenza-specific IgG, IgG1, and IgG2a. Samples were analyzed in triplicate. Serum was also titered for neutralizing antibodies by the HAI assay and SVN assay. (a) IgG. (b) IgG1. (c) IgG2a. (d) Serum HAI titres were determined using 0.5% red blood cells (RBCs) against the BC15 (H7N9) (WT) virus. The samples were analyzed in duplicate. The dotted line corresponds to the negative cut-off of 40, which is associated with a 50% reduction in the chance of contracting the influenza virus. (e) SVN titres were determined using 100 TCID₅₀/50 μL of the BC15 (H7N9) influenza virus. The samples were analyzed in quadruplicate. The data is shown as the mean ± the standard deviation. The dots correspond to the values obtained from individual mouse samples. Significant differences among groups are signified by * (p < 0.05), *** (p < 0.001) or **** (p < 0.0001). ns. = not significant. A one-way ANOVA was performed, which was followed by the Tukey post-hoc test to determine the p-value.

Figure 6

Antigen-specific IFN-γ and IL-5 secreting cells induced by QTV vaccination in mice. BALB/c mice were vaccinated with two doses of MEM (control) or QTV virus (1 × 10³ PFU). Nine days after the second vaccination (day 30), BALB/c mice (four mice per group, two males and two females) were euthanized and their splenocytes were isolated. The numbers of IFN-γ (a) and IL-5 (b) secreting T cells per 5 × 10⁵ splenocytes were determined by ELISPOT. Each sample was tested in triplicate. The antigen-induced counts were determined by subtracting the number of IFN-γ or IL-5 secreting cells with the medium-treated control cells. The data is shown as the mean ± the standard deviation. The dots correspond to the values obtained from individual mouse samples. Significant differences among groups are signified by **** (p < 0.0001). ns = not significant. A one-way ANOVA was performed, which was followed by the Tukey post-hoc test to determine the P-value.

Figure 7

The body weight, survival rate, and lung viral titration of vaccinated mice after viral challenge with the BC15 (H7N9) virus. BALB/c mice were intranasally vaccinated with two doses of MEM (control) or QTV virus (1 × 10³ PFU). These mice were then intranasally challenged with BC15 (H7N9) (WT) at a lethal dose of 1 × 10³ PFU. [n = eight per group, four males and four females for MEM / MEM and MEM / WT], [n = 10 per group, five males and five females for QTV / WT]. (a) The body weight. (b) The survival rate. (c) The viral titration of homogenized lung samples. Viral titration of the homogenized lung tissue was conducted by the TCID₅₀ assay. The samples were analyzed in quadruplicate. Four mice per group were humanely euthanized three days post-challenge, while the remaining mice were euthanized 14 days post-challenge. Dots correspond to the individual mouse lung titres. The four dots on three days post-challenge correspond to the four mice that were humanely euthanized. The one dot on six days post-challenge corresponds to one mouse that succumbed to infection. The three dots on seven days post-challenge correspond to three mice that succumbed to infection. The data is shown as the mean ± the standard deviation. The dots correspond to the values obtained from individual mouse samples.

Figure 8

Cytokine production after homologous viral challenge with BC15 (H7N9). The mRNA levels of RIG-I, IFN-α, IFN-β, IFN-γ, IP-10, TNFα, IL-6, IL-1β, IL-18, and IL-10 were determined from the mouse lungs harvested at three days post-challenge (n = four mice per group, two males and two females). These levels were assessed by qPCR, where each sample was tested in triplicate. The bars represent the mean value obtained from all mice in the respective group, and the error bar represents the standard deviation. Significant differences among groups are signified by ** (p < 0.01), *** (p < 0.001) or **** (p < 0.0001). ns. = not significant. A one-way ANOVA was performed, which was followed by the Tukey post-hoc test to determine the p-value.