Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus

Abstract

Background: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses.

Methods: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus.

Results: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge.

Conclusion: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

Keywords: Influenza virus; MVA vector; antibodies; vaccine.

Figure 1.

Western Blot analysis of HA and hlHA of CA/09 virus by recombinant MVA vectors. Protein expression and molecular weights were determined with an α-H1N1 chicken serum. Cell lysates from infected CEF (MOI 5) were analyzed 48 h p.i. Cell lysates from uninfected- and MVAwt-infected CEF were used as controls. Position and size (kDa) of molecular weight markers are indicated on the right side of each panel.

Figure 2.

Flow cytometry analysis of CEF infected by MVA-hlHA-CA/09 or by MVA-HA-CA/09 virus using three human monoclonal antibodies specific for stalk conformational epitopes (FB179, FE43, and FG20). Samples were treated with both primary antibody and FITC-labeled anti-human Ig-rabbit antibody (infected cells, red lines, and uninfected cells, black lines). As controls, infected cells were only treated with the second antibody (green lines).

Figure 3.

The different cellular distributions of full-length hemagglutinin (HA) (upper panel) and its headless derivative (hlHA) (lower panel) are shown by their co-localization with the ER marker calnexin (cnx). The headless derivative is largely retained in the ER, while the full-length HA localizes independently. The α-V5 antibody recognizes a tag at the C-terminus of both forms of the HA molecule.

Figure 4.

Serum antibody titers in mice following immunization with MVA-hlHA-CA/09 virus. Groups of C57BL/6 mice were immunized twice, three weeks apart, with MVA-hlHA-CA/09, MVA-HA-CA/09 or MVA-wt virus. (A) The titers of anti-influenza specific antibodies were determined with use of 50-fold-diluted samples obtained three weeks after the second immunization by titration on ELISA plates coated with recombinant HA proteins of CA/09, NC/99, Jap/57, VN/04, IN/05 or Bris/07 virus. Bars represent means ± standard deviation (SD) for six mice per group. OD 450 = Optical density at 450 nm. (B) Specific antibodies against CA/09 virus were measured in serum pools by HI and MN assays. ND = Not detectable.

Figure 5.

Vaccine-induced cellular immune responses in mice. Groups of mice were immunized twice with MVA-hlHA-CA/09 or MVA-wt virus. (A) Seven days later, freshly isolated splenocytes were cultured for 4 days in the presence of the recombinant HA protein from CA/09 virus, unloaded- or CA/09 virus loaded-APC. The proliferative response to Concanavalin A (Con A) was used as a control, and the fold increase in proliferation was calculated by determining the ratio of the stimulated:unstimulated proliferative index. (B) Four weeks later, recombinant HA protein was injected subcutaneously in the footpad and differences in footpad swelling between MVA-hlHA-CA/09- and MVAwt-vaccinated mice were determined 24 and 48 h after antigen injection by using a caliper. The data are the mean footpad swelling ± SD of three (for 24 h readings) and two (for 48 h readings) independent experiments. *P < 0.01, NS = Not significant, compared with MVAwt-immunized mice by unpaired Student’s t test.

Figure 6.

Effect of vaccination in influenza virus-challenged mice. Mice (13/group) were immunized as described in the legend for Figure 2, and challenged i.n. four weeks later with 3 LD₅₀ of CA/09 virus. (A) Seven days after challenge, mice (six/group) were sacrificed and IFN-γ production was measured by ELISPOT assay from bulk splenocytes and MLN of mice in the presence of recombinant HA protein from CA/09 virus or NP_366–374 peptide. Bars represent the mean values ± SD of triplicate cultures. *P < 0.0001 compared with MVAwt-immunized mice by unpaired Student’s t test. (B) Body weight and survival were monitored for fourteen days after virus infection and mice were sacrificed when body weight reached 75% of starting weight. The data shown are representative of three independent experiments.