Development of NP-Based Universal Vaccine for Influenza A Viruses

Abstract

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8⁺ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.

Keywords: adenoviral vector; autophagy; autophagy-inducing peptide; influenza vaccine; nucleoprotein; universal influenza vaccine.

Figure 1

(A) Diagrammatic image of HAd−C5−NP(H7N9), HAd−NP(H7N9), and HAd-ΔE1E3 genomic representation. The cytomegalovirus (CMV) promoter and the bovine growth hormone (BGH) polyadenylation signal are flanking the NP(H7N9) or C5-NP(H7N9) genes. The drawings are not to scale. LITR, left inverted terminal repeat; RITR, right inverted terminal repeat; ΔE1, deletion of E1 region; ΔE3, deletion of E3 region; C5, C5-AIP; NP, nucleoprotein. (B) Immunoblot confirming expression of NP(H7N9) or C5-NP(H7N9) in HAd-NP(H7N9)- or HAd-C5-NP(H7N9)-infected 293 cells, respectively. Mock or HAd-ΔE1E3 infected cell extracts were used as a negative control. The molecular weight marker is shown on the left. Outlines of the one-dose (C) or the two-dose (D) animal inoculation study.

Figure 2

Immunogenicity of HAd−NP(H7N9) or HAd−C5−NP(H7N9) in inoculated mice. BALB/c mice six to eight weeks old (5 animals/group) were vaccinated once intranasally (i.n.), as described in the material and methods section. After vaccination for four weeks, blood samples were collected and used to monitor the development of NP-specific IgG (A), IgG₁ (B), IgG_2a (C), and IgA (D) antibody responses by ELISA. Lung washes were also collected to monitor the development of mucosal NP-specific IgG (E), IgG₁ (F), IgG_2a (G), and IgA (H) antibody responses by ELISA. The data are displayed as the mean ± standard deviation (SD) of the optical density (OD). Enhancement in the number of NP-specific IFN-γ-secreting CD8 T cells following immunization with HAd-C5-NP(H7N9) was monitored at 4 weeks post-vaccination in the spleen (I), mediastinal lymph node (LN) (J), and lung mononuclear (MN) Cells (K) by enumerating NP-specific IFN-γ-secreting CD8 T cells by ELISpot using the NP-147 peptide. ns, non-significant at p > 0.05; *, significant at p < 0.05; **, significant at p < 0.01; ***, significant at p < 0.001; and ****, significant at p < 0.0001.

Figure 3

Protection efficacy of single i.n. vaccination of mice with Had-NP(H7N9) or Had-C5-NP(H7N9). At 4 weeks post-inoculation, immunized animal groups were challenged with 2 lethal doses of 50 (LD₅₀) of A/Puerto Rico/8/1934(H1N1) (A,B) or 5 LD₅₀ of A/Hong Kong/1/68(H3N2) (C,D). (A,C) Morbidity and (B,D) mortality after challenge were monitored. (E–G). Groups were challenged with 100 mouse infectious dose 50 (MID₅₀) of A/chukkar/MN/14951-7/1998(H5N2) (E), A/goose/Nebraska/17097/2011(H7N9) (F), or A/Hong Kong/1073/1999(H9N2) (G) influenza virus, and at 3 days post-challenge, the lungs were collected for virus titers. The data are shown as mean Log10 tissue culture infectious dose 50 (TCID₅₀) or egg infectious dose 50 (EID₅₀), and the detection limit was 0.5 Log10 TCID₅₀ or EID₅₀ per ml. ns, non-significant at p > 0.05; *, significant at p < 0.05; **, significant at p < 0.01; ***, significant at p < 0.001; and ****, significant at p < 0.0001.

Figure 4

Immunogenicity of Had-NP(H7N9) or Had-C5-NP(H7N9) two-dose regimen inoculation. BALB/c mice six to eight weeks old (5 animals/group) were immunized twice intranasally (i.n.), as mentioned in the material and methods section. Three weeks post-boost, blood samples were collected and used to monitor the development of NP-specific IgG (A), IgG₁ (B), IgG_2a (C), and IgA (D) antibody responses by ELISA. Three weeks post-boost, lung washes were also collected to monitor the development of mucosal NP-specific IgG (E), IgG₁ (F), IgG_2a (G), and IgA (H) antibody responses by ELISA. ELISA results are the optical density (OD) readings as mean ± standard deviation (SD). Enhancement in the number of NP-specific IFN-γ-secreting CD8 T cells following immunization with HAd-C5-NP(H7N9) was monitored at 3 weeks post-booster in the spleen (I), mediastinal lymph node (LN) (J), and lung mononuclear (MN) cells (K) by enumerating NP-specific IFN-γ-secreting CD8 T cells by ELISpot using the NP-147 peptide. ns, non-significant at p > 0.05; **, significant at p < 0.01; ***, significant at p < 0.001; and ****, significant at p < 0.0001.

Figure 5

Protection of Had-NP(H7N9) or Had-C5-NP(H7N9) two-dose regimen. At 3 weeks post-booster, immunized animal groups were challenged with 2 lethal doses of 50 (LD₅₀) of A/Puerto Rico/8/1934(H1N1) (A,B) or 5 LD50 of A/Hong Kong/1/68(H3N2) (C,D). (A,C) Morbidity and (B,D) mortality after challenge were monitored. (E–G). Groups were challenged with 100 mouse infectious dose 50 (MID₅₀) of A/chukkar/MN/14951-7/1998(H5N2) (E), A/goose/Nebraska/17097/2011(H7N9) (F), or A/Hong Kong/1073/1999(H9N2) (G) influenza virus, and the lungs were collected 3 days post-challenge and lung viral titers were determined. The data are shown as mean Log10 tissue culture infectious dose 50 (TCID₅₀) or egg infectious dose 50 (EID₅₀), and the detection limit was 0.5 Log10 TCID₅₀ or EID₅₀ per ml. ns, non-significant at p > 0.05; and ****, significant at p < 0.0001.

Figure 6

Histopathology of HAd−NP(H7N9)- or HAd−C5−NP(H7N9)-immunized mice lung tissues. (A) BALB/c mice (3 animals/group) were mock-immunized (PBS) or immunized intranasally (i.n.) with 10⁸ plaque-forming units (PFU) of HAd-∆E1E3, HAd-NP(H7N9), or HAd-C5-NP(H7N9). Animals were euthanized at 0.25-, 0.5-, 1-, 2-, 4-, and 8-day post-immunization, and the lung tissue samples were collected and processed for histopathology. Representative pictures of each group on days 1, 2, 4, and 8 post-immunization are shown (H&E, 200X). (B) The lung tissue histopathological scores from HAd-NP(H7N9)- or HAd-C5-NP(H7N9)-immunized mice. Lung tissue sections were blindly analyzed by a board-certified veterinary pathologist. ns, non-significant at p > 0.05; and *, significant at p < 0.05.

Figure 7

AIP-C5-mediated upregulation of genes involved in autophagy. BALB/c mice (3 animals/group) were mock-inoculated with PBS or inoculated i.n. once with 1 × 10⁸ PFU of Had-NP(H7N9), Had-C5-NP(H7N9), or HAd-ΔE1E3. The animals were euthanized at 24 h post-inoculation, and the lungs were collected for total RNA extraction. The RT² mouse autophagy PCR array was used to analyze the differentially expressed genes in all study groups. (A) The differentially expressed genes between the HAd-∆E1E3 and the PBS groups are presented as a volcano plot. (B) The differentially expressed genes between the HAd-NP(H7N9) and the PBS groups are presented as a volcano plot. (C) The differentially expressed genes between the HAd-C5-NP(H7N9) and the PBS groups are presented as a volcano plot. (D) A bar graph showing the biological pathways of the significantly upregulated gene list input of the HAd-C5-NP(H7N9) group over the PBS group, colored by the p-values, is presented using the Metascape web analysis tool. (E) The differentially expressed genes between the HAd-NP(H7N9) and the HAd-∆E1E3 groups are presented as a volcano plot. (F) The differentially expressed genes between the HAd-C5-NP(H7N9) and the HAd-∆E1E3 groups are presented as a volcano plot. (G) The differentially expressed genes between the HAd-C5-NP(H7N9) and the HAd-NP(H7N9) groups are presented as a volcano plot. (H) A bar graph showing the biological pathways of the significantly upregulated gene list input of the HAd-C5-NP(H7N9) group over the HAd-∆E1E3 group, colored by the p-values, is presented using the Metascape web analysis tool. (I) A dot blot chart of the gene ontology enrichment analysis for the biological pathways involved in the upregulated gene list of the HAd-C5-NP(H7N9) group using ShinyGO v0.76 is shown. (J) A heat map of the differentially expressed genes of the HAd-NP(H7N9) group and HAd-C5-NP(H7N9) group compared to the HAd-∆E1E3 group.