Omega-3 Polyunsaturated Fatty Acid: A Pharmaco-Nutraceutical Approach to Improve the Responsiveness to Ursodeoxycholic Acid.

Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.

Increased Immunogenicity of Full-Length Protein Antigens through Sortase-Mediated Coupling on the PapMV Vaccine Platform.

Background: Flexuous rod-shape nanoparticles-made of the coat protein of papaya mosaic virus (PapMV)-provide a promising vaccine platform for the presentation of viral antigens to immune cells. The PapMV nanoparticles can be combined with viral antigens or covalently linked to them. The coupling to PapMV was shown to improve the immune response triggered against peptide antigens (<39 amino acids) but it remains to be tested if large proteins can be coupled to this platform and if the coupling will lead to an immune response improvement. Methods: Two full-length recombinant viral proteins, the influenza nucleoprotein (NP) and the simian immunodeficiency virus group-specific protein antigen (GAG) were coupled to PapMV nanoparticles using sortase A. Mice were immunized with the nanoparticles coupled to the antigens and the immune response directed to the antigens were analyzed by ELISA and ELISPOT. Results: We showed the feasibility of coupling two different full-length proteins (GAG and NP) to the nanoparticle. We also showed that the coupling to PapMV nanoparticles improved significantly the humoral and the cytotoxic T lymphocyte (CTL) immune response to the antigens. Conclusion: This proof of concept demonstrates the versatility and the efficacy of the PapMV vaccine platform in the design of vaccines against viral diseases.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling.

BACKGROUND: Flexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles-a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated. RESULTS: We have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection. CONCLUSIONS: This technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

Engineering of the PapMV vaccine platform with a shortened M2e peptide leads to an effective one dose influenza vaccine.

The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6-14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can be an important component of a broadly cross-reactive influenza vaccine.