Gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous H5N1 infection

Abstract

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD(50)) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.

FIG. 1.

Characterization of BacHA. (A) Indirect immunofluorescence assay with insect cells infected with recombinant baculovirus expressing H5 HA0. Infected cells were fixed and stained with guinea pig anti-HA antibody and rabbit anti-guinea pig FITC. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified baculoviruses, showing the presence of major capsid protein (VP39) and envelope protein (GP64) of baculovirus and influenza HA in the purified sucrose gradient fraction. Lane 1, prestained protein marker; lane 2, infected cell culture supernatant; Lanes 3, 4, and 5, first, second, and third fractions from sucrose density gradient purification, respectively. (C) Western blot. The antigenic conformation of HA incorporated into purified baculoviruses (fraction 3) was probed using anti-HA monoclonal antibody.

FIG. 2.

Transduction and expression of HA in human intestinal epithelial cells by WSSV ie1-based BacHA. HCT116 cells were incubated with BacHA at a multiplicity of infection (MOI) of 200 and stained with anti-HA monoclonal antibody followed by FITC-conjugated secondary antibody at 20 h postinfection. Vero cells incubated with BacHA at the same MOI were used as a reference control. (A) Live BacHA-transduced HCT 116 cells. (B) HCT cells incubated with inactivated BacHA. (C) Live BacHA-transduced Vero cells. (D) Vero cells incubated with inactivated BacHA.

FIG. 3.

Measurement of systemic immune response. Groups of mice (n = 6) were orally vaccinated three times on days 0, 7, and 21 with 200 μl containing inactivated or live recombinant baculovirus at a log₂ HA titer of 8, adjuvanted with or without 10 μg rCTB. (A) HA-specific IgG antibody titers in serum, determined by indirect ELISA. (B) Serum hemagglutination inhibition titer. Each point represents the arithmetic mean value (n = 6) ± SD (***, P < 0.001).

FIG. 4.

Measurement of mucosal anti-HA specific IgA antibody levels by indirect ELISA. Groups of mice (n = 6) were orally vaccinated three times on days 0, 7, and 21 with 200 μl containing inactivated or live recombinant baculovirus at a log₂ HA titer of 8, adjuvanted with or without 10 μg rCTB. Each point represents the arithmetic mean value (n = 6) ± SD (*, P < 0.05; **, P < 0.02; ***, P < 0.001).

FIG. 5.

Cross-clade serum microneutralization in mice immunized orally with inactivated or live recombinant baculovirus at a log₂ HA titer of 8 with or without 10 μg rCTB. Viruses from clade 1.0 (A/Vietnam/1203/2004), clade 2.1 (A/Indonesia/CDC1031/2007), clade 2.3 (A/chicken/Nongkhai/NIAH400802/2007), and clade 8.0 (A/chicken/Henan/12/2004) were used for this study. Sera from the day of peak response, day 21 after the final immunization, were used for the assay. Each point represents the arithmetic mean value (n = 6) ± standard error (*, P < 0.05; **, P < 0.02; ***, P < 0.001).

FIG. 6.

Baculovirus transduction of mouse intestinal epithelial cells in vivo. One week after the third immunization, intestinal tissue samples were embedded in paraffin and sectioned. Immunohistochemical staining was carried out using a guinea pig anti-HA antibody and rabbit anti-guinea pig FITC. Intestinal villi of mice orally vaccinated with live baculovirus (A), inactivated baculovirus (B), and PBS (C) are shown.

FIG. 7.

Protection of mice from lethal H5N1 virus challenge. Groups of mice (n = 6) were orally vaccinated three times on days 0, 7, and 21 with 200 μl containing inactivated or live recombinant baculovirus at a log₂ HA titer of 8, adjuvanted with or without 10 μg rCTB. Wild-type baculovirus at a dose equivalent to BacHA (based on the viral titers determined by plaque assay) served as a negative control. (A and B) Three weeks after the final vaccination, mice were intranasally infected with 5 MLD₅₀ of homologous (CDC/669/Indonesia/06 clade 2.1) (A) and heterologous (Vietnam/1203/2004 clade 1.0) (B) HPAI H5N1 virus strains. Mice were monitored for survival throughout a 14-day observation period. The results are expressed in terms of percent survival. (C) The group of mice challenged with Vietnam/1203/2004 clade 1.0 was also monitored for weight loss throughout a 14-day observation period. The results are expressed as percent body weight (at the beginning of the trial).

FIG. 8.

Photomicrographs of hematoxylin- and eosin-stained lung sections of mice challenged with clade 2.1 H5N1 virus at 6 days postchallenge. (A) Mice vaccinated with live BacHA; (B) mice vaccinated with inactivated BacHA; (C) unvaccinated mice challenged with virus; (D) normal morphology seen in uninfected mice.