A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza

Protection of mice from lung viremia and serology results after <b>two</b> vaccinations.

(1)<p>hemagglutinin-inhibition titer determined with chicken erythrocytes;</p>(2)<p>geometric mean titer;</p>(3)<p>microneutralization assay (cpe-based);</p>(4)<p>neuraminidase-inhibition titer;</p>(5)<p>two separate experiments with 6 animals per group were performed;</p>(6)<p>inactivated whole virus vaccine, subcutaneous application route;</p>(7)<p>wild-type MVA (NIH74 LVD clone 6);</p>(8)<p>wild-type vaccinia Lister strain;</p>(9)<p>the dectection limit (dl) is <10;</p

T cell induction by the hemagglutinin expressing live vaccines in the lungs.

<p>Frequencies of influenza antigen specific IFN-γ+ CD4 T-cells (<b>A, C</b>) and CD8 T-cells (<b>B, D</b>) after immunizing two times with hemagglutinin constructs MVA-H1-Ca (<b>A, B</b>) or rVVL-H1-Ca (<b>C, D</b>) and stimulation with hemagglutinin (H1/CA-PP) or neuraminidase (N1/CA-PP) peptide pools. Lung cells or spleen cells were isolated at days 28, 41 and 45. Animals were challenged with wild-type swine flu virus on day 42, as indicated by the arrow. The filled (open) circles indicate lung (spleen) cells stimulated with the hemagglutinin peptide pool H1/CA PP. The filled (open) triangles indicate lung (spleen) cells stimulated with the neuraminidase peptide pool used as negative controls. Representative results of two independent experiments are shown.</p

Lung titers in Balb/c mice.

<p>The dots represent the lung titers of individual mice vaccinated with the different experimental vaccines or control preparations. Mice vaccinated with the controls (PBS, wild-type MVA (MVA wt) or Lister (VV-L wt) were not protected showing average log10 TCID50 titers of 5.2, 4.9 and 5.4, respectively. Mice vaccinated with inactivated vaccine (inact. H1N1), or two different doses (10⁶, 10⁷ pfu) of MVA-H1-Ca or rVVL-H1-Ca were close to or fully protected. Partial protection was achieved with two dosages of the MVA neuraminidase viruses (MVA-N1-Ca), while full protection was seen with the Lister-based neuraminidase virus (rVVL-N1-Ca). The dotted line represents the detection limit of log₁₀ 2.21. Low titers in range of log10<2.21 to 3.0 were confirmed by a passage assay of the lung samples in MDCK cells.</p

Geometric mean neutralization (NT) and hemagglutination inhibitions (HI) titers of mouse sera after single dose immunizations with the live vectors.

(1)<p>day 0 titers were all below the detection limit (dl);</p>(2)<p>wild-type MVA (NIH74 LVD clone 6); detection limits of the HI and the NT assay were <10.</p

T cell induction by the neuraminidase expressing live vaccines.

<p><b>A</b>) Frequencies of influenza antigen specific IFN-γ+ CD4 T cells after immunizing two times with MVA-N1-Ca (black bars), rVVL-N1-Ca (white bars) or inactivated vaccine (grey bars) and stimulation with buffer and with different antigens and peptides (shown on x-axis). Splenocytes were stimulated with protein antigens as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012217#pone-0012217-g004" target="_blank">Fig. 4</a>. <b>B</b>) Frequencies of influenza neuraminidase-specific IFN-γ+ CD8 T-cells after two dosages of vaccine. Splenocytes were stimulated with the peptide pools indicated in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012217#pone-0012217-g004" target="_blank">Fig. 4</a>. The data are mean values (+/− SEM) of two independent experiments.</p

Western blots of chicken cell (DF-1) lysates probed for influenza antigens.

<p>(A) Hemagglutinin expression in chicken cells. Lane 1, marker size in kDa. Lane 2, formalin-inactivated purified H1N1 influenza virus. Lane 3, negative control, DF-1 cells infected with wt vaccinia virus. Lane 4, replicating virus rVVL-H1-Ca, no trypsin treatment. Lane 5, replicating virus rVVL-H1-CA, with trypsin treatment. Lane 6, MVA-H1-Ca, no trypsin treatment. Lane 7, MVA-H1-Ca, with trypsin treatment. Lane 8, negative control lysate, MVA wt virus. Lane 9, uninfected cell lysate. HA0, unprocessed hemagglutinin; HA1 and HA2, the two processed HA subunits. B) Neuraminidase expression in chicken cells. Lanes 1-3, samples as above. Lane 4, replicating virus rVVL-N1-Ca. Lane 5, MVA-N1-Ca. Lane 6 and 7, negative controls, lysates of MVA wt virus and non-infected DF-1 cells. The band around 75 kDa represents the neuraminidase (NA).</p