Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

<div><p>The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.</p></div

Adoptive transfer of sera from rBac-HA-immune mice provides protection against challenge with heterosubtypic H5N1 virus.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera, BAL fluids and splenocytes collected from the immunized mice 3 weeks after second vaccination were adoptively transferred to naïve mice, and 24 h later these mice were challenged with 10 LD₅₀ of H5N1 influenza virus. Survival of recipient mice were monitored for 14 days after challenge. <i>Bac-ctrl</i>, Bac-control.</p

Construction and characterization of rBac-HA virus.

<p>(A) A schematic diagram of baculovirus vector construct containing HA gene. The pFastBac dual vector has engineered to encode pH1N1 HA gene from A/California/04/09 under the control of the polyhedrin (PH) promoter. The recombinant baculovirus, rBac-HA virus, was generated using the Bac-to-Bac baculovirus expression system. (B) The presence of multimeric HA protein in the purified viral particle was confirmed by cross-linking and western blotting as described in the Materials and Methods (HA proteins indicated by the arrowhead). Virus particles corresponding to 3×10⁶ PFU were cross-linked, heated in loading buffer at 65°C (cross-linked samples) or 100°C (non-cross-linked samples), and loaded for each lane. (C) The expression level of HA protein on the baculovirus envelope was analyzed using flow cytometry. The Sf9 cells were infected with rBac-HA virus or Bac-control at MOI of 10. 48 h post-infection, the cells were analyzed by HA-specific polyclonal antibody to determine the expression of rBac-HA on the envelope. Uninfected cells used as a negative control. <i>Bac-ctrl</i>, Bac-control-infected cells; <i>rBac-HA</i>, rBac-HA-infected cells; <i>Uninfected</i>, uninfected cells.</p

Sera, BAL fluids and splenocytes from rBac-HA virus-immune mice for adoptive transfer study.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera, BAL fluids and splenocytes were collected from the immunized mice 3 weeks after second vaccination. (A) Whole HA or HA stalk protein-specific IgG in the pooled sera and (B) HA-IgA or HA stalk-IgG in the pooled BAL fluids were measured by ELISA. The results indicate Log₂ end-point titers. (C) HA-specific CD8⁺ T cells (H-2K^d/HA_533-541 tetramer⁺, CD8⁺ and CD44⁺) were obtained from peripheral bood or lung tissue of boosted mice, and measured by flow cytometry. (D) Intracellular IFN-γ-producing CD8⁺ T cells (IFN-γ⁺, CD8⁺ and CD44⁺) from the donor lung tissue were measured by flow cytometry. “N.S.” indicates that there is no statistical significance between “PBS” group and “Bac-ctrl" group. *, Statistical significance to “Bac-control” (p<0.05). <i>N</i>.<i>S</i>., not significant; <i>Bac-ctrl</i>, Bac-control.</p

Mice immunized with rBac-HA virus show protection against challenge with heterosubtypic H5N1 virus.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁶ PFU or 3×10⁷ PFU of rBac-HA virus via intranasal route. Each group of mice was challenged by intranasal administration with 50 LD₅₀ of H5N1 influenza virus. (A) Weight loss was monitored daily for 14 days after challenge. The results were expressed in percent body weight compared to beginning of the trial. (B) Survival of all group of mice were also monitored for 14 days. *, Statistical significance with “Bac-ctrl” group (p<0.05). <i>Bac-ctrl</i>, Bac-control.</p

rBac-HA virus elicited stalk-specific Abs.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁶ PFU or 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera were collected from all mice 3 weeks after second vaccination. HA stalk-specific Abs were measured in boosted mice by ELISA using chimeric H9/1 protein [H9 HA head on top of an H1 (PR8) stalk domain]. The result was expressed absorbance at 450 nm. <i>O</i>.<i>D</i>., optical density; <i>Positive</i>, Sera from mice vaccinated with 3×10⁷ PFU of recombinant adenovirus encoding HA of H5N1 virus and ectodomain of matrix 2 protein (M2e) of H1N1 virus; <i>Bac-ctrl</i>, Bac-control.</p

Humoral immune response induced by intranasal rBac-HA virus immunization.

<p>BALB/c mice (n = 4/group) were immunized twice on day 0 and 16 with 1×10⁶ PFU or 3×10⁶ PFU of rBac-HA virus via intranasal route. Sera were collected from primed and boosted mice 2 and 3 weeks after vaccination, respectively. Control mice were immunized i.n. with 3×10⁶ PFU of Bac-control. The group of mice injected with PBS as negative control. (A) HA-specific and (B) Baculovirus-specific IgG antibody titers were measured in primed and boosted mice sera by ELISA, respectively. (C) Hemagglutination inhibition (HAI) titers against A/California/04/09 were measured in the sera obtained from boosted mice. (D) Mucosal HA-specific IgA in BAL fluid were measured 3 weeks after vaccination by ELISA. The results indicate Log₂ end-point titers. “N.S.” indicates that there is no statistical significance between “PBS” group and “Bac-ctrl" group. *, Statistical significance with “Bac-ctrl” group (p<0.05). ^‡, Statistical significance with “Priming” group (p<0.05). ^†, Statistical significance with “PBS” group (p<0.05). <i>N</i>.<i>S</i>., not significant; <i>Bac-ctrl</i>, Bac-control.</p