A novel M2e based flu vaccine formulation for dogs.

BACKGROUND: The USA 2004 influenza virus outbreak H3N8 in dogs heralded the emergence of a new disease in this species. A new inactivated H3N8 vaccine was developed to control the spread of the disease but, as in humans and swine, it is anticipated that the virus will mutate shift and drift in the dog population. Therefore, there is a need for a vaccine that can trigger a broad protection to prevent the spread of the virus and the emergence of new strains. METHODOLOGY AND PRINCIPAL FINDINGS: The universal M2e peptide is identical in almost all the H3N8 influenza strains sequenced to date and known to infect dogs. This epitope is therefore a good choice for development of a vaccine to provide broad protection. Malva mosaic virus (MaMV) nanoparticles were chosen as a vaccine platform to improve the stability of the M2e peptide and increase its immunogenicity in animals. The addition of an adjuvant (OmpC) purified from Salmonella typhi membrane in the vaccine formulation increased the immune response directed to the M2e peptide significantly and enlarged the protection to include the heterosubtypic strain of influenza in a mouse model. An optimal vaccine formulation was also shown to be immunogenic in dogs. CONCLUSIONS AND SIGNIFICANCE: The MaMV vaccine platform triggered an improved immune response directed towards the universal M2e peptide. The adjuvant OmpC increased the immune response to the M2e peptide and protection to a heterosubtypic influenza strain that harbors a different M2e peptide in a mouse model. Antibodies generated by the vaccine formulation showed cross-reactivity with M2e peptides derived from influenza strains H9N2, H5N1 and H1N1. The vaccine formulation shows a potential for commercialization of a new M2e based vaccine in dogs.

Engineering of papaya mosaic virus (PapMV) nanoparticles with a CTL epitope derived from influenza NP.

BACKGROUND: The ever-present threat of infectious disease, e.g. influenza pandemics, and the increasing need for new and effective treatments in immunotherapy are the driving forces that motivate research into new and innovative vaccine platforms. Ideally, such platforms should trigger an efficient CTL response, be safe, and easy to manufacture. We recently developed a novel nanoparticle adjuvant comprised of papaya mosaic virus (PapMV) coat protein (CP) assembled around an RNA. The PapMV nanoparticle is an efficient vaccine platform in which the peptide antigen is fused to the C-terminus of the PapMV CP, leading to nanoparticles presenting surface-exposed epitope. The fusion stabilizes the epitope and improves its immunogenicity. We found recently that C-terminal fusions are not always efficient, depending on the nature of the peptide fused to the platform. RESULTS: We chose a CTL epitope derived from the nucleocapsid (NP) of influenza virus (NP147₋155) for this proof-of-concept demonstration. Recombinant nanoparticles harbouring a fusion at the N-terminus were more efficient in triggering a CTL response. Efficacy appeared to be linked to the stability of the nanoparticles at 37°C. We also showed that discs--smaller than nanoparticles--made of 20 subunits of PapMV CP are less efficient for induction of a CTL response in mice, revealing that assembly of the recombinant PapMV CP into nanoparticles is crucial to triggering an efficient CTL response. CONCLUSION: The point of fusion on the PapMV vaccine platform is critical to triggering an efficient CTL response. Efficacy is linked to nanoparticle stability; nanoparticles must be stable at 37°C but remain susceptible to cellular proteases to ensure efficient processing of the CTL epitope by cells of the immune system. The results of this study improve our understanding of the PapMV vaccine platform, which will facilitate the design of efficient vaccines to various infectious threats.

Engineering of papaya mosaic virus (PapMV) nanoparticles through fusion of the HA11 peptide to several putative surface-exposed sites.

Papaya mosaic virus has been shown to be an efficient adjuvant and vaccine platform in the design and improvement of innovative flu vaccines. So far, all fusions based on the PapMV platform have been located at the C-terminus of the PapMV coat protein. Considering that some epitopes might interfere with the self-assembly of PapMV CP when fused at the C-terminus, we evaluated other possible sites of fusion using the influenza HA11 peptide antigen. Two out of the six new fusion sites tested led to the production of recombinant proteins capable of self assembly into PapMV nanoparticles; the two functional sites are located after amino acids 12 and 187. Immunoprecipitation of each of the successful fusions demonstrated that the HA11 epitope was located at the surface of the nanoparticles. The stability and immunogenicity of the PapMV-HA11 nanoparticles were evaluated, and we could show that there is a direct correlation between the stability of the nanoparticles at 37°C (mammalian body temperature) and the ability of the nanoparticles to trigger an efficient immune response directed towards the HA11 epitope. This strong correlation between nanoparticle stability and immunogenicity in animals suggests that the stability of any nanoparticle harbouring the fusion of a new peptide should be an important criterion in the design of a new vaccine.

Improvement of the PapMV nanoparticle adjuvant property through an increased of its avidity for the antigen [influenza NP].

The principal caveat of existing influenza vaccine is their failure to provide long-term protection. This lack of efficiency is caused by persistent (drift) and dramatic (shift) antigenic changes on the major surface proteins, the main target of protective immunity generated by traditional vaccines. Alternatively, vaccination with most conserved protein, like the nucleoprotein (NP) can stimulate immunity against multiple serotypes and could potentially provides an extended protection. The NP antigen contains more than 90% protein sequence homology among influenza A isolates and it also contains dominant CTL targets epitopes that made this antigen an attractive target for developing universal vaccine. However, NP protein is a weak antigen and need the use of adjuvant to increase its immunogenicity. We have developed an innovative high avidity VLP (HAV) nanoparticle to improve its adjuvant property to the NP antigen. The nanoparticles are derived from papaya mosaic virus capsid protein (PapMV CP) produced in a bacteria expression system. We generated the HAV by adding an affinity peptide directed to the NP protein at the surface of the VLPs. The fusions of the affinity peptide to PapMV VLPs increased the avidity of PapMV VLPs to NP protein. This modification enhanced the humoral and the IFN-γ response directed to NP. Moreover, the immunity generated by the HAV adjuvanted NP vaccine improved the protection of vaccinated mice to a challenge with influenza virus. The protection was characterized by accelerated virus elimination after the onset of infection and rapid recovery of the vaccinated animals.