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Obtaining the recombinant N protein of the RSV has faced critical problems of purity and solubility. Jean-François Eléouët and collaborators have solved these difficulties when they have developed and patented an original method for production and purification of the N protein based on the procaryotic co-expression of said protein with the truncated P protein of the RSV. Afterwards, the N protein is easily isolated from these soluble complexes.

The N protein forms subnucleocapsid ring structures -SRS (10 nm of diameter; homogenous rings of 10 N proteins enclosing a 70b RNA), which have showed their immunogenic properties and vaccine potential in the murine model. Nasal administration of NSRS protein, with or without the mucosal adjuvant LT(R192G) (detoxified E. coli enterotoxin), has induced a strong production of antibodies in the serum. Interestingly, NSRS protein has stimulated the mucous secretion of specific IgA, even in absence of the dedicated adjuvant. Moreover, the cellular immune responses (CD4 and CD8 mediated T cell responses) were also stimulated at a local and systemic level by the nasal delivery of NSRS with adjuvant. Experimental infection of vaccinated animals has showed a significant decrease of the viral replication associated with a moderate inflammation (neutrophils) in the lung tissue. The vaccine potential of the NSRS was also evaluated the calf model of the RSV infection. The vaccination protocol (intra-muscular priming and boost) of the animals reared in isolation units was followed by an infectious challenge. The NSRS vaccine led to a partial protection of calves against the disease and virus replication, a strong humoral immunogenicity (systemic and nasal anti-N antibodies) and memory cellular response (sub-maxillary lymph node), without any sign of disease exacerbation.

The team of Jean-François Eléouët has also showed that NSRS protein can be used as a support to graft heterologous proteins. Soluble complexes of N protein-GFP (Green Fluorescent Protein) were produced and purified as subnucleocapsid ring structures. Vaccination of mice with NSRS-GFP has induced an increased antibody response against GFP compared to administration of the GFP alone. Confocal microscopy showed that NSRS-GFP complexes were adsorbed and captured by various cell types having potential activities in immune responses (dendritic cells, monocytes, epithelial cells), demonstrating that the N protein of the RSV can be used as a carrier for molecules of interest, such as therapeutic or vaccine ones. This strategy could be implemented for the development of the RSV vaccine, thanks to the presentation on NSRS of additional epitopes of the viral enveloppe glycoproteins, known to induce neutralizing antibodies.

POTENTIAL INDUSTRIAL APPLICATION

The patented method for production of the nucleocapsid N protein of Paramyxoviridae allows the development of various industrial applications in human health and in the veterinary field:

• Vaccination against Paramyxoviridae, including the human RSV and the bovine RSV
• Development of diagnostic tools targeting Paramyxoviridae
• Use of the N subnucleocapsid ring structures as carriers for molecules of interest, such as therapeutic or vaccine proteins

Patents: WO2006/117456 et WO2007/119011

Publications:

• Tran TL, Castagné N, Bhella D, Varela PF, Bernard J, Chilmonczyk S, Berkenkamp S, Benhamo V, Grznarova K, Grosclaude J, Nespoulos C, Rey FA and Eléouët JF.
  The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer. Journal of General Virology (2007), 88, 196–206

• Roux X, Dubuquoy C, Durand G, Tran-Tolla TL, Castagné N, Bernard J, Petit-Camurdan A, Eléouët JF, Riffault S.
  Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus. PLoS ONE 3(3): e1766