Interferon mediated prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine lacking non-structural protein 1

Abstract

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Figure 1

Pre-incubation with ΔNS1 virus inhibits viral replication in embryonated chicken eggs. (A) 10-day-old embryonated chicken eggs (n = 2 per group) were inoculated with varying amounts of (PFU) of ΔNS1 virus in the allantoic cavity. Eight hours post infection at 37 °C, eggs were re-infected with 10⁴ PFU of WT A/WSN/33 influenza virus and incubated at 37 °C for 40 h. Allantoic fluids were then titrated by plaque assay MDBK cells. (B) 10-day-old embryonated chicken eggs (n = 2 per group) were inoculated with 2 × 10⁴ PFU of ΔNS1 virus or PBS (Untreated). 8 h post inoculation at 37 °C, the eggs were re-infected with 10³ PFU of A/WSN/33 (WSN/H1N1), A/PR/8 (PR8/H1N1), A/X-31 (X-31/H3N2), B/Lee/40 (B-Lee influenza B) or Sendai Virus (Sendai). B-Lee infected eggs were incubated at 35 °C for additional 40 h. All other eggs were incubated at 37 °C for additional 40 h. Virus present in the allantoic fluid was titrated by hemagglutination assays. Maximum hemagglutination titers (100%) for each individual virus were 2048 (PR8), 1024 (X-31), 256 (B-Lee), 512 (Sendai).

Figure 2

A single dose of ΔNS1 virus protects A2G mice against lethal infection by highly virulent hvPR8 influenza virus when given prior to virus challenge. (A) Treatment with ΔNS1 virus protects A2G mice against lethal infection by highly virulent hvPR8 influenza virus. Eight 6-week old A2G mice were intranasally infected with 5 × 10⁶ PFU of highly virulent A/PR/8/34 (hvPR8) influenza virus. Half of the mice received a total of five intranasal treatments with 5 × 10⁵ PFU of ΔNS1 virus at the following times with respect to the hvPR8 infection: − 24 h, − 8 h, + 3 h, + 24 h ad 48 h. The remaining four mice were treated with PBS and the bodyweight changes and survival was monitored. (B) A single dose of ΔNS1 virus protects A2G mice against lethal infection by highly virulent hvPR8 influenza virus when given prior to hvPR8 virus challenge. Groups of three A2G mice each were mock-treated or treated intranasally with 5 × 10⁵ PFU of ΔNS1 at time points − 24 h, − 8 h, + 3 h, + 24 h, + 48 h relative to the intranasal infection by 5 × 10⁶ hvPR8 influenza virus. (C) A single dose of ΔNS1 virus protects A2G mice against lethal infection by highly virulent hvPR8 influenza virus when given 2 and 4 days prior to hvPR8 virus administration Groups of three A2G mice were intranasally treated with 5 × 10⁵ PFU of ΔNS1 virus 4 days or 2 days before infection by 5 × 10⁶ hvPR8 influenza virus. Bodyweight changes and survival was monitored. All data points are from individual mice. (D) Determination of the minimal effective therapeutic dose of ΔNS1 to prevent lethal hvPR8 virus infection in A2G mice. Groups of three A2G mice were intranasally infected with 10⁵, 10⁴ or 10³ PFU ΔNS1 influenza virus. Additionally, groups of two A2G mice were intranasally challenged with 10² of ΔNS1 virus or PBS. 24 h post inoculation, mice were challenged with by 5 × 10⁶ hvPR8 influenza virus. The percentage of mice surviving the challenge is represented.

Figure 3

Dose dependent pre-treatment of ΔNS1 protects A2G-Mx1 mice but not wild-type C57BL/6 from a lethal hvPR8 virus challenge. (A) Induction of Mx1 specific mRNA expression in ΔNS1 virus infected mice. Groups of two A2G or BALB/c mice were intranasally treated with PBS or 2.5 × 10⁵ PFU of ΔNS1 hvPR8 influenza viruses. 24 h post challenge, total RNA present in lung tissues were extracted and were used for RT-PCR reactions using Mx1 specific primers. PCR products were run in an agarose gel; the arrows indicate the predicted size of amplified cDNA from Mx1 genes pf A2G mice (Mx1) and BALB/c mice (Mx1*). (B, C, D, E) Sex matched 6 weeks old groups C57BL/6-A2G-Mx1 mice or C57BL/6-wild-type mice were either intranasally pre-treated with PR8-ΔNS1 (5 × 10⁶ PFU; n = 5 per group), sterile PBS (n = 5) 12 h before a lethal challenge of hvPR8 (5 × 10⁵ PFU; n = 5) or treated with only sterile PBS (n = 2). (B) Morbidity of C57Bl/6-A2G-Mx1 mice. (C). Survival of C57Bl/6-A2G-Mx1 mice. (D). Morbidity of C57Bl/6-wild-type mice. (E). Survival of C57Bl/6–6-wild-type mice.

Figure 4

Comparison of the antiviral properties in A2G mice of recombinant influenza A viruses ΔNS1 and D2. A2G mice were intranasally treated with PBS or 2.5 × 10⁵ PFU of ΔNS1 or D2 viruses for 24 h before infection with 5 × 10⁶ PFU of hvPR8 influenza virus. Bodyweight changes and survival were monitored. Data shown are from individual mice.

Figure 5

Treatment with ΔNS1 influenza virus protects C57BL/6 mice against lethal infection with Sendai virus. All mice were challenged intranasally with a lethal dose of Sendai virus corresponding to (A) 5 × 10⁵ PFU or (B) 1.5 × 10⁵ PFU. The percentage of mice surviving the challenge is represented. (A) Groups of five mice were treated intranasally with 2.5 × 10⁵ PFU of ΔNS1 virus at the indicated times. (B) Groups of five mice were intranasally treated at − 24 h and + 24 h with respect to the infection with Sendai virus with 2.5 × 10⁵ PFU of ΔNS1 or with the indicated amounts of IFN-β.

Figure 6

Treatment with ΔNS1 influenza virus inhibits viral replication in the lungs of K18-hACE2 mice challenged with SARS-CoV-2. Mice were intranasally treated with 30 ul containing PBS, 2.5 × 10⁶ PFU of ΔNS1, 1 μg defective interfering RNA from Sendai virus (SDI-RNA), 2.5 × 10⁵ U of universal-interferon (uIFN) 24 h before intranasal challenge with 10⁴ PFU of SARS-CoV-2/USA/WA1 isolate. (A) Weight-loss was monitored in mice (n = 11 for treated groups and n = 6 naïve) and (B) survival was monitored for 12 days. (C) Lungs were harvested at days three and five post infection (n = 3 per group per day) were homogenized and were titered in Vero-E6 cells using standard plaque assays.