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. 2014 Feb 11;9(2):e88340.
doi: 10.1371/journal.pone.0088340. eCollection 2014.

MVA vectors expressing conserved influenza proteins protect mice against lethal challenge with H5N1, H9N2 and H7N1 viruses

Annett Hessel  1 , Helga Savidis-Dacho  2 , Sogue Coulibaly  2 , Daniel Portsmouth  1 , Thomas R Kreil  3 , Brian A Crowe  1 , Michael G Schwendinger  1 , Andreas Pilz  1 , P Noel Barrett  1 , Falko G Falkner  4 , Birgit Schäfer  1
Affiliations

MVA vectors expressing conserved influenza proteins protect mice against lethal challenge with H5N1, H9N2 and H7N1 viruses

Annett Hessel et al. PLoS One. .

Abstract

Background: The availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes.

Methods: Mice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively.

Results: MVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4(+) and CD8(+) T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4(+) T cell responses and NP-specific CD8(+) T cell responses were associated with increased protective efficacy.

Conclusions: MVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4(+) and CD8(+) T cell responses.

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Conflict of interest statement

Competing Interests: AH, BS, HS-D, SC, DP, TRK, BAC, MGS, AP, PNB and FGF report being employed by Baxter. HS-D, SC, TRK, BAC, MGS, PNB and FGF report having an equity interest in the company. AH, BS, TRK, PNB and FGF report a submitted patent on MVA vectors: “Recombinant viral vectors and methods for inducing a heterosubtypic immune response to influenza A virses” WO 2012/106231; PCT/US2012/023085. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Scheme of HA stem and HA stem/M2e sequence.
(A) Schematic representation of the HA stem (hlHA) and HA stem/M2e constructs, depicting i) the hemagglutinin (HA) of A/Vietnam/1203/2004 H5N1 (VN1203) from which the hlHA and hlHA/M2e constructs were derived; ii) the HA stem (hlHA) construct which contains the HA signal sequence, the HA1 amino acids (AA) 17–58 and 290–343, which are linked by four glycine (G) residues, the polybasic cleavage site, and the HA2 sequence; iii) the HA stem/M2e fusion protein which contains the M2e domains of H5N1, H1N1, H9N2, H7N2, linked to each other by a GSAGSA linker, and linked to the HA1 sequences by three glycine (G) residues. (B) Schematic representation depicting the nucleotide (nt) positions within the MVA vector into which conserved influenza proteins are inserted.
Figure 2
Figure 2. Weight loss after H5N1, H9N2 and H7N1 challenge.
Mice were immunized twice, three weeks apart, with MVA vectors and challenged three weeks later with (A and B) 42 LD50 of H5N1 VN1203, (C) 32 LD50 of mouse-adapted H9N2 HK/G9 or (D) 16 LD50 of H7N1 RO/34. (B) Primed mice were infected intranasally with 100 TCID50 H1N1pdm09 virus, six weeks before immunization. Animals were monitored for 14 days after challenge. Shown are the daily variations in weight, as percentages compared to before virus challenge.
Figure 3
Figure 3. Symptom scores after H5N1, H9N2 and H7N1 challenge.
Mice were immunized twice, three weeks apart, with MVA vectors and challenged three weeks later with (A and B) 42 LD50 of H5N1 VN1203, (C) 32 LD50 of mouse-adapted H9N2 HK/G9 or (D) 16 LD50 of H7N1 RO/34. (B) Primed mice were infected intranasally with 100 TCID50 H1N1pdm09 virus, six weeks before immunization. Animals were monitored for 14 days after challenge. Shown are the cumulative mean symptom scores whereby ruffled fur, curved posture, apathy and death were scored as 1, 2, 3 and 4, respectively.
Figure 4
Figure 4. Antibody and T cell responses.
(A) ELISA titers against whole-virus H5N1. The starting dilution of the assay is 1∶100. (B) Percentage of CD4+ T cells reacting to whole-virus H5N1, H1N1 and H9N2 and to recombinant H5 hemagglutinin (rHA H5) antigen after immunization of mice with MVA vectors. (C) Percentage of CD8+ T cells reacting to HA and NP peptides. Asterisks denote statistical significance calculated by two-way ANOVA and Bonferroni posttests (*p<0.01, **p<0.001, ***p<0.0001).
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This study was funded by Baxter. Baxter employees were responsible for study design, data collection and analysis, decision to publish, and preparation of the manuscript.