Protein and modified vaccinia virus Ankara-based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice

Abstract

Because of the high variability of seasonal influenza viruses and the eminent threat of influenza viruses with pandemic potential, there is great interest in the development of vaccines that induce broadly protective immunity. Most probably, broadly protective influenza vaccines are based on conserved proteins, such as nucleoprotein (NP). NP is a vaccine target of interest as it has been shown to induce cross-reactive antibody and T cell responses. Here we tested and compared various NP-based vaccine preparations for their capacity to induce humoral and cellular immune responses to influenza virus NP. The immunogenicity of protein-based vaccine preparations with Matrix-M™ adjuvant as well as recombinant viral vaccine vector modified Vaccinia virus Ankara (MVA) expressing the influenza virus NP gene, with or without modifications that aim at optimization of CD8⁺ T cell responses, was addressed in BALB/c mice. Addition of Matrix-M™ adjuvant to NP wild-type protein-based vaccines significantly improved T cell responses. Furthermore, recombinant MVA expressing the influenza virus NP induced strong antibody and CD8⁺ T cell responses, which could not be improved further by modifications of NP to increase antigen processing and presentation.

Keywords: MVA; Matrix-M™; adjuvant; immunogenicity; influenza virus; nucleoprotein; vaccine.

Figure 1

Nucleoprotein (NP)‐specific antibody responses after NP wild‐type (NPwt) protein (with or without Matrix‐M™ adjuvant) or recombinant Modified Vaccinia virus Ankara (rMVA)–NPwt vaccination. The immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses 21 days after the primary vaccination (a,b) or 14 days after the booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean of each group is indicated. MM = Matrix‐M™ adjuvant. ****P < 0·0001.

Figure 2

Vaccination with wild‐type nucleoprotein (NPwt) protein with Matrix‐M™ adjuvant or recombinant Modified Vaccinia virus Ankara (rMVA)–NPwt induced NP‐specific splenocyte responses. Spleens obtained 14 days after the booster vaccination were stimulated with purified NPwt protein. The number of interleukin (IL‐)2 (a), interferon (IFN‐)γ (b) and IL‐2/IFN‐γ (c)‐producing T cells was used to determine NP‐specific splenocyte activation in spot‐forming units (SFU)/10⁶ cells. Samples were tested in triplicate. Mean of each group is indicated. MM = Matrix‐M™ adjuvant. *P < 0·0306; **P < 0·0082; ***P = 0·0005.

Figure 3

Nucleoprotein (NP)‐specific antibody responses induced by vaccination with NP wild‐type (NPwt) protein + Matrix‐M^TM adjuvant or the respective recombinant Modified Vaccinia virus Ankara (rMVA)–NP constructs. Immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses in mice 21 days after the primary vaccination (a,b) or 14 days after booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean for each group is indicated. MM = Matrix‐M™ adjuvant. *P < 0·0432; **P < 0·0068; ***P < 0·0009; ****P < 0·0001.

Figure 4

Vaccine‐induced nucleoprotein (NP)‐specific T cell responses. (a–c) Splenocytes were stimulated with the NP_147–155 peptide after which the number of interleukin (IL‐)2 (a), interferon (IFN‐)γ (b) or IL‐2/IFN‐γ (c) spot‐forming units (SFU)/10⁶ splenocytes were determined. Samples were measured in triplicate. The mean for each group is indicated. (d) Splenocytes were stimulated with the NP_147–155 peptide for 12 h. Using flow cytometry, live splenocytes were selected and subsequently CD3⁺, CD8⁺ and IFN‐γ⁺ cells were gated. Representative graphs of mice vaccinated with wild‐type Modified Vaccinia virus Ankara (wtMVA) as a control or recombinant MVA (rMVA)–NPwt are shown. (e) IFN‐γ production of CD3⁺CD8⁺ splenocytes per 10⁶ events measured using flow cytometry. Mean for each group is indicated. MM = Matrix‐M^TM adjuvant. *P < 0·0227; **P = 0·0073; ***P = 0·0005; ****P < 0·0001.