A new self-attenuated therapeutic influenza vaccine that uses host cell-restricted attenuation by artificial microRNAs

Abstract

New strategies are urgently needed for developing vaccines and/or anti-viral drugs against influenza viruses, because antigenic shift and drift inevitably occurs in circulating strains each year, and new strains resistant to anti-viral drugs have recently emerged. In our study, we designed and incorporated artificial microRNAs (amiRNAs) into the NA segment of rescued influenza viruses to separately target two host genes, Cdc2-like kinase 1 (CLK1) and SON DNA binding protein (SON), which were found to play an essential role in virus replication. Mouse epithelial fibroblast (MEF) or human lung carcinoma A549 cells infected with engineered influenza PR8 viruses expressing amiR-30CLK1 (PR8-amiR-30CLK1) or amiR-93SON (PR8-amiR-93SON) had reduced expression of host proteins CLK1 and SON, respectively. All engineered influenza viruses functioned as attenuated vaccines, induced significantly higher antibody responses, and provided greater protective efficacy. In addition, they were found to be safe, based on the mouse weight changes and clinical signs observed. In contrast to the engineered viruses targeting SON, mice treated with engineered viruses targeting CLK1 recovered from weight loss and survived lethal infection by 6 h after lethal-dose PR8 infection, suggesting that our PR8-amiR-30CLK1 self-attenuated influenza virus (SAIV) could be used as a new therapeutic influenza vaccine.

Keywords: Artificial microRNA; Cdc2-like kinase 1; Intranasal delivery; Live attenuated influenza vaccine; SON DNA-binding protein; Therapeutic vaccine.

Figure 1.. Design of artificial miRNAs (amiRs) based on human miR-30 and miR-93 backbones and detection of CLK1 and SON transcripts.

(A) Sequence and secondary structure of human miR-30. (B) Sequence and secondary structure of amiR-30CLK1. (C) 293T cells were transfected with pll3.7, pll3.7-miR-30, or pll3.7-amiR-30CLK1. Forty-eight hours later, total RNA was extracted and reverse-transcribed, the CLK1 cDNA amplified by PCR, and the products separated by electrophoresis on an agarose gel, with GAPDH used as the control. (D) Sequence and secondary structure of human miR-93. (E) Sequence and secondary structure of amiR-93SON. (F) 293T cells were transfected with pll3.7, pll3.7-miR-93, or pll3.7-amiR-93SON. Forty-eight hours later, total RNA was extracted and reverse-transcribed, the SON cDNA amplified by PCR, and the products separated by electrophoresis on an agarose gel, with GAPDH used as control.

Figure 2.. Engineering of the NA segment and verification of rescued influenza viruses.

(A) Diagrams of the original and engineered NA segments. Blue bars represent 3’ and 5’ noncoding regions (NCRs), and red bars represent the NA coding sequence. (Top) Organization of the original NA segment. (Bottom) Organization of the engineered NA segment with the miRNA- or amiRNA-expressing cassette. (B) Viral RNAs were isolated from PR8-wt, PR8-miR-30, PR8-amiR-30CLK1, PR8-miR-93, or PR8-amiR-93SON, and 1 μg of RNA was separated on a 4% acrylamide gel in TBE with urea for silver staining to detect influenza gene segments. (C) Viral RNAs were reverse-transcribed and then amplified and separated by electrophoresis on an agarose gel.

Figure 3.. CLK1 and SON expression in MEF or A549 cells infected with wild-type or engineered PR8 viruses.

MEF (top) or A549 cells (bottom) were infected with wild-type or engineered PR8 viruses. Forty-eight hours later, the cells were collected, total RNA extracted and reverse-transcribed, and the proteins extracted to examine CLK1 expression. (A) CLK1 segments were amplified by PCR and separated on an agarose gel. (B) SON segments were amplified by PCR and separated on an agarose gel. (C) CLK1 proteins were detected by western blotting.

Figure 4.. Antibody responses induced by wild-type or engineered PR8 viruses in mouse blood.

BALB/c mice (6–8 weeks old) in each group (n=10) were intranasally inoculated with 3×10⁵ PFU engineered viruses, 25 PFU wild-type PR8 (as positive control), or PBS (as negative control). Blood was collected on days 0, 7, 14, and 21 post inoculation. Concentrations of IgA, IgG, IgG1, and IgG2a antibodies were measured by ELISA. Comparisons among groups were performed by using a nonparametric one-way ANOVA with the Tukey multiple comparison test. The bars represent the mean ± standard error of the mean (SEM). P values <0.05 were considered to indicate a significant difference. As described, letter combinations above the bars indicate significant differences between groups, whereas shared letters indicate no significant difference [41].

Figure 5.. Protection induced by engineered or low-titer (25 PFU) wild-type PR8 viruses against wild-type PR8 challenge in mice.

See Fig. 4 legend for group and inoculation description. On day 21 post inoculation, mice were challenged with 50×MLD₅₀ and were then monitored daily for clinical symptoms, weight loss, and death for 21 days. Mice undergoing a weight loss in excess of 30% were euthanized for reasons of animal welfare. Body weight changes (A) were expressed as a percentage of baseline values. The black lines indicate mean values, and error bars represent the SEM. The t-test was used to compare body weight changes. The log-rank test was performed to establish significant differences between survival curves (B). P values < 0.05 were considered to indicate a significant difference. *significant difference in survival rates between groups.

Figure 6.. Therapeutic effects of engineered PR8 viruses.

BALB/c mice (6–8 weeks old) in each group (n=10) were infected with 20 × MLD₅₀ (10⁴ PFU) wild-type PR8. Six hours later, mice were intranasally dosed with 3×10⁵ PFU of engineered PR8 viruses. Mouse weight changes (A) and survival rates (B) were recorded for 21 days. See Fig. 5 legend for data description and statistical analysis.