Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice
Introduction
Influenza is a highly contagious viral respiratory disease that causes significant morbidity and mortality worldwide each year (de Jong and Hien, 2006). Current pandemic situation with new H1N1 swine-origin influenza A virus (S-OIV) has stressed the urgent need for safe and effective vaccines (CDCP, 2009). At present, influenza vaccines are generally administered via subdermal or intramuscular route to stimulate systemic immune response to prevent the disease (Ruat et al., 2008, Gao et al., 2006). However, for infection acquired through respiratory system, mucosal immune response plays an important role in protecting the host at the port of entry (Cox et al., 2004). Mucosal immunization, which is shown to stimulate both mucosal and systemic immune responses, would be an effective way to control the infection by influenza viruses (Ogra et al., 2001). Despite the recent attention towards intranasal administration to enhance mucosal immune responses, oral vaccination is still considered as the most effective way to increase patient compliance (Mann et al., 2004, CDCP, 2004). Non-invasive, pain-free self-administration, affordability, improved logistics and mass coverage during pre-pandemic and pandemic situations make oral vaccination an attractive option.
Several studies have previously reported the use of inactivated whole-virus, split or subunit antigens to study the efficacy of oral immunization against influenza (Amorij et al., 2007, Bender et al., 1996). Though some of these vaccines have proven to be effective, the production of these vaccines still possess some safety and technical issues as most method still relies on the culture of live viruses in embryonated chicken eggs. Moreover, the current method of producing influenza vaccines is technically challenging owing to the constraints in using high level biocontainment facilities and thorough inactivation procedures. Moreover, inactivated whole-virus and split-virus vaccines were shown to activate CD81 cytotoxic T-lymphocyte responses only rarely, have poor cross-reactivity to antigenic variants, and produce poor secretory immunoglobulin A (IgA) responses (Barackman et al., 2001).
Baculovirus expression system has long been used to produce recombinant proteins due to the proper post-translational modifications and high yield in insect cells (He et al., 2009). HA protein produced in the baculovirus expression system has been extensively evaluated in humans as influenza vaccines (Treanor et al., 2007). However, influenza HA expressed in insects cells is highly hydrophobic and its low solubility increases the difficulty of purification reducing its effectiveness as vaccines (Treanor et al., 2001). Baculovirus surface display technology has recently evolved as a novel platform for vaccine development against influenza viruses. This system enables the presentation of large complex proteins on the surface of baculovirus particles in their native functional conformation (Peralta et al., 2007), resulting in superior immune response when used as immunogens. More importantly, baculovirus is naturally replication-incompetent in mammalian cells (Tani et al., 2003) and even do not require the use of any live influenza viruses during vaccine development, manufacturing and administration processes. As an alternative to conventional egg-based influenza vaccines, the baculovirus expression system exploits serum-free insect cell suspension culture resulting in simplified cultivation and purification procedures. Combining the advantages of both baculovirus surface display technology and oral immunization for the development of influenza vaccines will have the added advantage of safety and immunogenecity.
The only major obstacle associated with the oral immunization approach is that the antigen in the vaccine formulation can be substantially degraded by gastric hydrochloric acid and proteolytic enzymes present in the gut (Pauletti et al., 1996), resulting in poor immunogenicity. Hence, in the present report, we have described a novel approach to deliver baculovirus displaying HA into the gastrointestinal tract of the mice using a reverse micelle-based carrier vehicle. This strategy will entrap the antigen within a particulate structure and prevents them from being exposed to the destructive gut components. We have also assessed the effect of BacHA/vaccine oil formulation with rCTB as mucosal adjuvant.
Section snippets
Influenza viruses
The highly pathogenic influenza A Human H5N1 viruses such as CDC/669/Indonesia/06 (GeneBank accession no. CY014481), CDC/594/Indonesia/06 (GeneBank accession no. CY014272) were obtained from Ministry of Health (MOH), Indonesia. The H5N1 viruses from different phylogenetic clades were rescued by Reverse Genetics (WHO, 2005). Briefly, the hemagglutinin (HA) and neuraminidase (NA) genes of H5N1 viruses from clade 1.0 (A/Vietnam/1203/2004), clade 2.1 (A/Indonesia/CDC1031/2007, clade 4.0
Structural and antigenic conformation of baculovirus surface displayed HA0
Confocal microscopic analysis indicated that HA0 expressed by the recombinant baculovirus was able to successfully translocate to the plasma membrane of infected insect cells (Fig. 1A). Further western blot analysis and hemagglutination assay demonstrated that baculovirus surface displayed HA0 was able to sustain its authentic cleavage (Fig. 1B) and hemagglutinin activity (data not shown).
Systemic antibody response to the oral delivery of En-BacHA
Mice immunized orally with En-BacHA showed significantly enhanced HA specific serum IgG titers when
Discussion
Development of vaccination strategies that effectively induce mucosal immunity would be of major interest for preventing influenza infection. Previous studies have already reported the feasibility of oral vaccination to induce mucosal immune response (IgA) in the respiratory tract to confer protection against influenza viruses (Pang et al., 1992, Bender et al., 1996). Mucosal IgA responses have also been shown to exhibit cross-protective immunity against antigenically distinct viruses (Liew et
Acknowledgements
The authors are grateful for the financial support received from Temasek Life Science Laboratory, Singapore. The authors thank the Ministry of Health (MOH), Indonesia for technical support and collaboration. The authors thank Dr. Ruben Donis, Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA, for providing the plasmids for reverse genetics. The authors also thank Hui Ting Ho for generation of RG-H5N1 (clade 1.0) virus.
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Both authors contributed equally to this work.