Preliminary Study On Influenza A Virus-like Particle Vaccine

Influenza is one of the most serious respiratory diseases which causes significant morbidity and mortality worldwide. The pandemic virus of swine-derived influenza A (H1N1) continues to cause infection. As of 18 July, 2010, WHO has reported at least 18,336 fatal cases in more than 214 countries. Meanwhile, highly pathogenic avian influenza (HPAI) H5N1 virus has resulted in 310 fatal cases since 2003, it has presented a high mortality rate of approximately 59%. Vaccine is the best way for preventing influenza. Conventional split seasonal influenza vaccine can provide effective protection against influenza but still have drawbacks. First, the manufacture process of conventional influenza vaccines relies on the growth of embryonated chicken eggs, which is labor intensive and slow, would not be able to meet demand during an influenza pandemic. There are not ideal influenza vaccines for infants, elderly and hypersensitivity patients. Second, Error-prone RNA-dependent RNA polymerase and segmented genome permit influenza viruses to mutate (antigenic drift) and recombinant (antigenic shift) frequently and enable the viruses to escape aggression by immune system. As derived from prototype viruses and aiming to induce antibody against two major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), current influenza vaccines should update yearly. As a result, the fast production of influenza vaccines which provide cross-protection against different influenza variants or strains is highly desirable.Several studies have focused on developing non-replicating virus-like particles (VLPs). The first human vaccine manufactured using recombinant DNA technology was the enveloped hepatitis B vaccine produced in yeast, which has been used for over a decade. HPV-VLPs produced in yeast or insect cells were highly immunogenic resulting in successful clinical trials for preventing HPV infection and FDA approval for human use. As an alternative to conventional egg-based influenza vaccine approaches, recombinant-based influenza VLPs vaccine candidates produced with a baculovirus system are promising, innovative technology for efficient, safe, high-yielding and low-cost, influenza VLPs have been developed by several laboratories and were demonstrated to induce protective immunity in animal studies. The M2 protein is a type III integral membrane protein of influenza virus. It was first discovered by Lamb et al. The extracellular N-terminal domain of M2 protein (M2e) is highly conserved across human influenza A subtypes and provides a potential antigen target for developing a universal influenza vaccine with broad-spectrum prevention. Since M2e antigen is a 23 amino acids peptide, researchers focus on converting a normally non-immunogenic M2e-peptide into a highly immunogenic antigen. Several strategies were used to increase the immunogenicity of M2e peptide, such as increasing the peptide density, fusing M2e with large molecular weight carrier. Hepatitis B core protein (HBc), a structural protein of Hepatitis B virus, is one of the most frequent used carriers for delivering foreign epitopes. The common form of HBc protein is spherical particle with 120 spikes assembled by 240 metamers. Spikes, also called major immunodominant region (MIR), can present 240 copies of foreign epitopes on the surface of chimeric core particles and augment specific B-cell and T-cell immunogenicity significantly. M2e peptide inserted into C-terminally truncated HBc protein is highly immunogenic, induces an anti-M2e antibody titer higher than anti-HBc antibody titer and provides effective protection against a lethal viral challenge to mice. Moreover, HBc virus-like particles is efficiently produced in E. coli and can be obtained easily as a pure form.Fears of human to human pandemic H5N1 virus request M2e-based universal influenza vaccine provide subtype cross-protection against unexpected avian strains. But there are at least five different amino acids between human-type M2e and avian-type M2e sequences. Fan et al demonstrated that serum antibodies induced by human-type M2e fail to react with the M2e peptide of H5N1 virus in ELISA assay. It may reduce the efficiency of human-type M2e-based universal vaccine for resisting avian influenza. Internal influenza antigens, such as nucleoprotein (NP), are highly conserved among all influenza A subtypes, which can provide protection in animal studies by meditating subtype cross-reactive T-cell reaction. However, Heinen et al reported that a universal vaccine candidate contained nucleoprotein aggravates disease after challenge with influenza virus. So choosing suitable epitope of NP may decrease the risk of vaccine and facilitate fused HBc protein to form chimeric virus like particles (VLPs). The cytotoxic T lymphocytes (CTL) epitope aa418-426 of NP from H5N1 and other influenza virus can cross-recognized by human influenza CTL. Therefore, NP418-426 epitope, as a potential complement of human-type M2e-based vaccine, is likely to make the vaccine more efficient for preventing a future avian influenza outbreak.Based on the background above, the research was carried out in two parts:In the first part, we produced 2009 H1N1 virus-like particle (VLP) comprised Hemagglutinin (HA), Neuraminidase (NA) and Matrix 1 (M1) protein of 2009 H1N1 influenza virus in insect cells by Bac-to-Bac baculovirus expression systems. Purified 2009 H1N1 VLP was characterized by hemagglutination assay, negative staining electron microscopy, single-radial-immunodiffusion assay and western blot, the results showed that 2009 H1N1 VLP morphologically resembled influenza virions and exhibited biological characteristics of 2009 H1N1 influenza virus. 4-6 weeks old BALB/c mice were vaccinated with purified 2009 H1N1 VLP and then challenged with 2009 H1N1 virus. Results of ELISA and hemagglutination inhibition assay showed that 2009 H1N1 VLP could obviously elicit strong immune responses than split vaccinated groups. In addition, 2009 H1N1 VLP provide protective immunity in mice against the 2009 pandemic H1N1 influenza virus. All the results suggest that 2009 H1N1 VLP can be an excellent influenza vaccine that should be developed further.In the second part, two forms of fusion vaccine candidates were constructed: (1) fusing the NP418-426 epitope to the C-terminus of three tandem copies of human-type M2e and inserting the chimeric peptide antigen into MIR of C-terminally truncated HBc, designated 3M2e-NP-HBc; (2) inserting the same three tandem copies of M2e into MIR of HBc-N149 and designated 3M2e-HBc. We evaluated whether the fused NP418-426 epitope will make human-type M2e-based universal vaccine provide additional subtype cross-protection against the challenge of HPAI H5N1 virus. Both 3M2e-NP-HBc and 3M2e-HBc particles could greatly elicit high titers of M2-especific IgG antibody in vaccinated mice. Fused NP418-426 epitope enhanced cellular immune responses in 3M2e-NP-HBc vaccinated group than 3M2e-HBc vaccinated group, as indicated by higher titers of IgG2a and more IFN-γ-secreting lymphocytes. 3M2e-NP-HBc was more effective than 3M2e-HBc in resisting A/Ostrich/SuZhou/097/2003 (H5N1) virus and the 3M2e-NP-HBc vaccinated group could reach a 90% survival rate under 10LD₅₀ lethal dosage of H5N1 virus challenge. Our results indicated that chimeric HBc particle vaccine candidate 3M2e-NP-HBc not only provided full protection against novel H1N1 influenza virus but also enhanced protection against H5N1 avian influenza virus, suggesting that the new formulation of universal influenza vaccine candidate could be developed further as a broad-spectrum vaccine.