Generation of an Attenuated Chimeric Bat Influenza A Virus Live-Vaccine Prototype

Abstract

Recurring epizootic influenza A virus (IAV) infections in domestic livestock such as swine and poultry are associated with a substantial economic burden and pose a constant threat to human health. Therefore, universally applicable and safe animal vaccines are urgently needed. We recently demonstrated that a reassortment-incompatible chimeric bat H17N10 virus harboring the A/swan/Germany/R65/2006 (H5N1) surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) can be efficiently used as a modified live influenza vaccine (MLIV). To ensure vaccine safety and, thus, improve the applicability of this novel MLIV for mammalian usage, we performed consecutive passaging in eggs and chickens. Following passaging, we identified mutations in the viral polymerase subunits PB2 (I382S), PB1 (Q694H and I695K), and PA (E141K). Strikingly, recombinant chimeric viruses encoding these mutations showed no growth deficiencies in avian cells but displayed impaired growth in human cells and mice. Homologous prime-boost immunization of mice with one of these avian-adapted chimeric viruses, designated rR65_mono/H17N10_EP18, elicited a strong neutralizing antibody response and conferred full protection against lethal highly pathogenic avian influenza virus (HPAIV) H5N1 challenge infection. Importantly, the insertion of the avian-adaptive mutations into the conventional avian-like A/SC35M/1980 (H7N7) and prototypic human A/PR/8/34 (H1N1) viruses led to attenuated viral growth in human cells and mice. Collectively, our data show that the polymerase mutations identified here can be utilized to further improve the safety of our novel H17N10-based MLIV candidates for future mammalian applications. IMPORTANCE Recurring influenza A virus outbreaks in livestock, particularly in swine and chickens, pose a constant threat to humans. Live attenuated influenza vaccines (LAIVs) might be a potent tool to prevent epizootic outbreaks and the resulting human IAV infections; however, LAIVs have several disadvantages, especially in terms of reassortment with circulating IAVs. Notably, the newly identified bat influenza A viruses H17N10 and H18N11 cannot reassort with conventional IAVs. Chimeric bat influenza A viruses encoding surface glycoproteins of conventional IAV subtypes might thus function as safe and applicable modified live influenza vaccines (MLIVs).

Keywords: bat influenza A virus; influenza A virus; modified live influenza vaccine.

FIG 1

Serial passaging of R65_mono/H17N10 in eggs and day-old chicks. Recombinant R65_mono/H17N10 generated on HEK293T cells and subsequently amplified on MDCK II cells was repeatedly passaged (10 times) in 9- to 11-day-old eggs. Virus-containing allantoic fluid was subsequently used to infect day-old chicks (chicken passage [CP]). Virus-positive organ material from the conchae was then consecutively passaged eight times in 11- to 18-day-old eggs (embryonic passage 18 [EP18]). Finally, virus stocks were propagated in MDCK II cells. Deep sequencing of the total vRNA was performed for the passages indicated at the top. Mutation variant frequencies of >90% are highlighted in boldface type.

FIG 2

Mutations acquired upon passaging in 18-day-old eggs and day-old chicks impair viral growth in mammalian cells and mice. (A) To determine the impact of the mutations acquired upon passaging in day-old chicks and eggs, we generated recombinant R65_mono/H17N10 viruses harboring the amino acid substitutions PB2_I382S and PA_E141K (rR65_mono/H17N10_CP) or the two PB1 substitutions Q694H and I695K (rR65_mono/H17N10_EP18). Parental rR65_mono/H17N10 served as the control. Human A549 (top) and avian DF-1 (bottom) cells were infected at a multiplicity of infection (MOI) of 0.01. The virus supernatant was harvested at the indicated time points, and viral titers were determined via a plaque assay. (B) Groups of C57BL/6 mice (n = 5) were intranasally infected with 5 × 10³ PFU of the indicated viruses in 40 μL, and organ viral titers were determined at 3 dpi via a plaque assay. The dashed lines indicate the detection limit. Data are shown as means ± standard deviations (SD) from ≥3 experiments; statistical analysis was performed using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Insertion of the avian-adaptive mutations into A/SC35M/1980 and A/PR/8/34 affects viral growth in human cells. (A) To investigate the mutational impact in the context of conventional IAV isolates, we generated recombinant A/SC35M/1980 viruses comprising either PB2_I382S and PA_E141K (rSC35M_CP) or the two PB1 substitutions Q694H and I695K (rSC35M_EP18). rSC35M served as the wild-type control. Human A549 (top) and avian DF-1 (lower) cell lines were infected at an MOI of 0.001. The virus supernatant was collected at the indicated time points, and viral titers were determined via a plaque assay. The dashed lines indicate the detection limit. (B) Avian LMH and human HEK293T cells were transfected to reconstitute the SC35M polymerase harboring the avian-adaptive mutations PB2_I382S and PA_E141K or PB1_Q694H/I695K. At 24 h posttransfection, viral polymerase activity was measured and normalized to the wild-type polymerase activity, which was set to 100% (indicated by the dashed lines). (C and E) The viral growth of SC35M (C) and A/PR/8/34 (E) viruses coding for PB2_I382S or PA_E141K was evaluated on A549 (top) and DF-1 (bottom) cells infected at an MOI of 0.001. The virus supernatant was collected at the indicated time points, and viral titers were determined via a plaque assay. (D and F) Polymerases of SC35M (D) and PR8 (F) harboring the PB2_I382S or PA_E141K substitution were reconstituted in LMH and HEK293T cells. At 24 h posttransfection, viral polymerase activity was measured and normalized to the wild-type polymerase activity, which was set to 100% (indicated by the dashed lines). Data are shown as means ± SD from 3 experiments; statistical analysis was performed using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

PB2_I382S and PA_E141K attenuate viral replication of PR8 and SC35M viruses in mice. Groups of C57BL/6 mice (n = 5) were intranasally infected with 5 × 10³ PFU of SC35M (A) or 5 × 10² PFU of PR8 (B) viruses in 40 μL, and organ viral titers were determined at 3 dpi via a plaque assay. The dashed lines indicate the detection limit. Data are shown as means ± SD statistical analysis was performed using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Avian-adapted rR65_mono/H17N10_EP18 is a versatile platform to generate MLIVs. (A) To evaluate the transmission potential of the MLIV, groups of C57BL/6 mice (n = 3) were inoculated with 5 × 10³ PFU of rR65_mono/H17N10 or rR65_mono/H17N10_EP18 in 40 μL intranasally. At 1 dpi, infected index mice were cohoused with naive contact mice (n = 5) for 3 days. Organ viral titers were determined at 4 dpi via a plaque assay. MLIV protective efficacies were determined by immunizing groups of mice with 1 × 10⁶ PFU of either rR65_mono/H17N10 or rR65_mono/H17N10_EP18 in 40 μL using a homologous prime-boost vaccine strategy. Mock (PBS)-treated animals served as the controls. Three weeks after the boost, mice were challenged with 3× LD₅₀ of A/swan/Germany/R65/2006 (H5N1). (B) Mice of each group (n = 5) were sacrificed to determine organ viral titers of the A/swan/Germany/R65/2006 challenge virus at 3 dpi via a plaque assay. (C) Survival and changes in the body weights of the groups (n = 7) were monitored for 14 days. Mice were sacrificed and scored dead when 75% of their initial body weight was reached (dashed line). (D and E) Neutralizing antibodies against A/swan/Germany/R65/2006 (H5N1) (D) and A/Thailand/1 (Kan-1)/2004 (E) in the sera of vaccinated and unvaccinated mice were analyzed via a PRNT₅₀ assay.