Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1

Abstract

An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)-forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×10(7) FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.

Fig 1. Schematic diagrams of recombinant baculoviruses.

Diagram of the AcHERV-HA baculovirus containing HERV env and HA genes, the AcHERV-VLP virus additionally including NA and M1 genes under the control of the polyhedron promoter and CMV promoter, respectively. The recombinant baculoviruses were generated using the Bac-to-Bac baculovirus expression system.

Fig 2. Characterization of the recombinant baculoviruses (rBV; AcHERV-HA and AcHERV-VLP) and their expression in mammalian cells.

(A) PCR detection of HERV, HA, NA, and M1 genes in constructed baculovirus DNA. Lane 1: control for PCR; lane 2: AcHERV-HA baculovirus DNA; lane 3: AcHERV-VLP baculovirus DNA. (B) Western blot analysis of HA protein expression in recombinant baculovirus (rBV)-infected 293T cells. Mock, AcMNPV-infected cells; AcHERV-HA, AcHERV-HA–infected cells; AcHERV-VLP, AcHERV-VLP–infected cells. (C) Immunofluorescence micrograph of rBV-infected 293T cells. Seventy-two hours after infection, the cells were incubated with a mouse antibody against pH1N1, followed by incubation with a FITC-conjugated goat anti-mouse IgG antibody. Mock, AcMNPV-infected cells; AcHERV-HA, AcHERV-HA–infected cells; AcHERV-VLP, AcHERV-VLP–infected cells; Merge, merged image. (D) Transmission electron microscopy of negatively stained, purified VLPs from rBV-infected 293T cells. Seventy-two hours after infection, VLPs were harvested from 293T cells, concentrated, and partially purified using 20% step sucrose gradient ultracentrifugation. For electron microscopy, VLPs were stained with 1% uranyl acetate. Scale bar, 100 nm (× 100,000) or 200 nm (× 120,000). Wild-type pH1N1 purified A/California/04/2009 influenza virus; AcHERV-HA, purified VLP from AcHERV-HA–infected 293T cells; AcHERV-VLP, purified VLP from AcHERV-VLP–infected 293T cells.

Fig 3. Serological analysis in mice immunized with killed vaccine and baculoviruses.

Sera from mice injected intramuscularly with PBS, killed vaccine, AcHERV-HA, or AcHERV-VLP were collected and evaluated for immune response. (A) Antigen-specific IgG antibody titers against pH1N1 in mouse sera (on days 0, 7 and 21) were determined by ELISA. A450 refers absorbance at 450 nm. (B) HAI titer against the pH1N1 strain or PR8 strain in mouse sera on day 21. (C) Neutralizing titer against the pH1N1 strain or PR8 strain in mouse sera on day 21. ELISA and HAI assays were performed using ten randomly selected samples from each group (10/12). Neutralizing assays were performed using eight randomly selected samples from each group (8/12). Values in parentheses indicate number of mice tested/total number of mice immunized in each group. All experiments were run in triplicate. The data shown are means ± SEM for samples. Statistical analysis showed that data were significant with p<0.05 or not significant (one way ANOVA and two-tailed Student’s t-test): killed vaccine and AcHERV HA groups were compared with AcHERV VLP group (on days 21).

Fig 4. IFN-γ production in mice immunized with killed vaccine and baculoviruses.

Two weeks after the final immunization, splenocytes from mice injected intramuscularly with PBS, killed vaccine, AcHERV-HA, or AcHERV-VLP were collected and evaluated for the number of IFN-γ secreted spots from pH1N1-specific T cells in splenocytes. ELISPOT assays were performed using two randomly selected samples from each group (2/12). Values in parentheses indicate number of mice tested/total number of mice immunized in each group. All experiments were run in triplicate. The data shown are actual number of IFN-γ spot as a scatter dot and mean value as a line. Statistical analysis showed that data were significant with p<0.05 (ANOVA and two-tailed Student’s t-test): killed vaccine and AcHERV HA groups were compared with AcHERV VLP group.

Fig 5. Protective effect of immunization against challenge with a lethal dose of ma-pH1N1.

Body weights of mice intranasally challenged with a 20LD₅₀ dose of ma-pH1N1 2 weeks after the final immunization were monitored for 14 consecutive days. (A) Percent body weight change after challenge with a 20LD₅₀ dose of ma-pH1N1. Changes in body weight (n = 10 mice/group) are expressed as the mean value for each group. (B) Survival rate after challenge with a 20LD₅₀ dose of ma-pH1N1. Statistical analysis performed between the AcHERV-HA group and AcHERV-VLP group. Statistical analysis showed that data were significant with *p < 0.05 (two-tailed Student’s t-test).

Fig 6. Histological lesions in lung sections from immunized mice after challenge with pH1N1.

A subset of mice (n = 2 mice/group) from each group was sacrificed 4 days post challenge, and their lungs were H&E-stained for histological evaluation. (A) Non-infected BABL/c mice; (B) mice injected with PBS; (C) mice vaccinated with killed vaccine; (D) mice vaccinated with AcHERV-HA; (E) mice vaccinated with AcHERV-VLP. Arrows indicate the infiltration of inflammatory cells, including infiltration in vessels, the pulmonary parenchyma, and alveolar septa. Scale bar, 100 μm.