Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

Abstract

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.

FIG 1

Genetic organization of DNA vaccines based on PB1, NP, and M1 in pSCA vector and the resulting protein expression in MDCK cells transfected with recombinant plasmids. (A) Schematic diagram of the genetic organization of the pSCA DNA vector (top). The bottom shows the cDNA fragments of influenza viruses BJ95 PB1, NP, and M1 flanked by BamHI, BamHI, and SmaI restriction sites, respectively. CMV, cytomegalovirus. (B) Indirect immunofluorescence showing the expression of influenza PB1, NP, and M1 in MDCK cells transfected with pSCA-PB1 (left), pSCA-NP (middle), and pSCA-M1 (right) recombinant plasmids (top) stained with the polyclonal or monoclonal antibodies (Abs) indicated in Materials and Methods. The bottom images show the results of MDCK cells mock transfected with pSCA plasmid and detected using the same Abs as in the top images.

FIG 2

Genetic organization of PB1-, NP-, and M1-expressing pJSA1175 recombinant plasmids containing homologous sequences from vaccinia virus and the resulting protein expression in HeLa cells and CEFs infected with recombinant vaccinia virus. (A) Schematic diagram of the genetic organization of the pJSA1175 DNA vector (top). The bottom shows the cDNA fragments of influenza virus BJ95 (H3N2) PB1, NP, and M1 after restriction digestion with BamHI or SmaI, as appropriate, and smoothing, respectively. (B) Indirect immunofluorescence showing the expression of influenza PB1, NP, and M1 in plaques formed in rVV-PB1 (left)-, rVV-NP (middle)-, and rVV-M1 (right)-infected HeLa cells (upper) and stained with the polyclonal or monoclonal Abs indicated in Materials and Methods. The bottom images show the results of HeLa cells mock infected with rVV and analyzed using the same Abs as in the top images. (C) Western blots showing the expression of influenza PB1, NP, and M1 from rVV-PB1 (lane 1)-, rVV-NP (lane 2)-, and rVV-M1 (lane 3)-infected CEF cell lysates stained with the polyclonal or monoclonal antiserum indicated in Materials and Methods. M, molecular weight marker; Neg, cell lysates from CEFs mock infected with rVV-c.

FIG 3

Humoral and cellular immune responses induced by influenza virus PB1-, NP-, and M1-based vaccines. Mice were immunized intramuscularly with influenza virus PB1-, NP-, and M1-based DNA or rVV vaccines, according to the immunization schedule described in Table 1. (A) Humoral immune response in influenza virus PB1, NP, and M1 vaccine-immunized mice. A serum sample was obtained from each mouse at week 8, and the presence of IgG antibodies specific for influenza A virus was analyzed using ELISA. The bars show the geometric mean antibody titers, and the error bars indicate 95% confidence intervals (n = 14 mice/group). (B) Cellular immune responses in influenza virus PB1, NP, and M1 vaccine-immunized mice. Mice were sacrificed at week 8, and the spleens were separated under aseptic conditions and ground to isolate SMNCs (n = 3 mice/group). Next, 4 μg/ml NP_147–155 (TYQRTRALV) (B1) and PB1_317–325 (MFLAMITYI) (B2) were used as stimulants in ELISPOT assays. The numbers of SMNCs that produced IFN-γ after stimulation with peptides for 30 h are presented as spot-forming cells (SFCs)/10⁶ SMNCs. The bars show mean SFCs/10⁶ SMNCs, and the error bars indicate standard deviations. Lines above two or more groups indicate comparable results. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using one-way ANOVA.

FIG 4

Protective efficacy of influenza A virus PB1, NP, and M1 vaccines against 1.7 LD₅₀ of PR8. Fifteen groups of mice were immunized with the vaccine control (A), NP (B), PB1 (C), M1 (D), or a combination of the NP, PB1, and M1 (E) DNA, rVV, or prime-boost DNA-rVV vaccines (Table 1) at the times indicated in Fig. 3. Mice were challenged with 1.7 LD₅₀ of influenza virus PR8 at week 8 and were monitored daily for 17 days after challenge (n = 10 mice/group). The mice were weighed daily to detect morbidity (left graphs). The mean weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight was calculated based on the mean weight of each group at day 0. The survival rates (right graphs) were calculated following challenge.

FIG 5

Protective efficacy of immunization with influenza virus PB1-, NP-, and M1-based vaccines against 5 LD₅₀ of PR8. Fifteen groups of mice were immunized with the vaccine control (A), NP (B), PB1 (C), M1 (D), or a combination of the NP, PB1, and M1 (E) DNA, rVV, or prime-boost DNA-rVV vaccines (Table 1) at the times indicated in Fig. 3. Mice were challenged with 5 LD₅₀ of influenza virus PR8 at week 8 and monitored daily for 17 days after challenge (n = 10 mice per experimental group). The mice were weighed daily to detect morbidity (left graphs). The mean weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight was calculated based on the mean weight of each group at day 0. The survival rates (right graphs) were calculated following challenge.

FIG 6

Protective efficacy of immunization with influenza virus NP-based vaccines against 10 LD₅₀ of PR8. Nine groups of mice were immunized with the vaccine control (A), NP (B), or a combination of the NP, PB1, and M1 (C) DNA, rVV, or prime-boosted DNA-rVV vaccines (Table 1) at the times indicated in Fig. 3. Mice were challenged with 10 LD₅₀ of influenza virus PR8 at week 8 and monitored daily for 17 days after challenge (n = 10 mice/group). The mice were weighed daily to detect morbidity (left graphs). The mean weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight was calculated based on the mean weight of each group at day 0. The survival rates (right graphs) were calculated following challenge.