Engineering of papaya mosaic virus (PapMV) nanoparticles with a CTL epitope derived from influenza NP
© Babin et al.; licensee BioMed Central Ltd. 2013
Received: 14 November 2012
Accepted: 25 March 2013
Published: 4 April 2013
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.
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.
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.
Papaya mosaic virus (PapMV) is a member of the large family of Flexiviridae in the genus Potexvirus. The virus has a flexuous rod shape of 500 nm in length and 13 nm in diameter . The CP, made mostly of alpha helices , is composed of 215 amino acids and has an estimated molecular weight of 23 kDa . We showed previously that non-infectious nanoparticles made of recombinant PapMV CP are similar in shape and appearance to wild-type virus purified from plants . PapMV nanoparticles were used previously as a vaccine platform technology to improve the immunogenicity of a peptide antigen fused to the nanoparticle structure [4–8]. The PapMV vaccine platform can induce a long-lasting memory response to an antigen fused on its surface . Previous studies showed the capacity of PapMV nanoparticles to trigger a CTL response, in both in vitro and in vivo models, when the CTL epitope was fused to the C-terminus of the CP [6, 7, 9]. Although PapMV tolerates insertion of several peptides to its C-terminus [4–7, 10], a recent study revealed that N-terminal fusion of some peptides is also tolerated . Depending on the nature of the amino acid sequence, some peptides can interfere with the CP assembly or with nanoparticle stability, which can affect their ability to stimulate an humoral response. A modification of the fusion site on the CP can help to resolve this issue. In this study, we compared the efficacy of nanoparticles harbouring fusion of a CTL epitope at either the N- or the C-terminus to trigger a cellular immune response.
The crystalline and highly ordered structure of the nanoparticles is critical to triggering an efficient humoral response, as also reported by many other groups [11–13]. However, it is still unknown if assembly into the highly ordered nanoparticle structure, made of several hundreds of PapMV CP, is more efficient than assembly of a smaller disc-like structure (aggregate of 20 subunits) in triggering the CTL response. Since the mechanisms of induction of humoral and CTL immune responses rely on different immune cells and mechanisms, we also evaluated the importance of highly ordered assembly of recombinant PapMV CP into nanoparticles in triggering a CTL response by comparing the immunogenicity of nanoparticles and discs.
Results and discussion
Engineering of PapMV nanoparticles fused to the influenza CTL epitope
We previously reported that residue F13 of PapMV CP is critical for the interaction between the PapMV CP subunits when assembling into nanoparticles . We showed that this hydrophobic residue fits snugly inside the hydrophobic pocket of the neighbouring CP . Interestingly, insertion of the NP147-155 epitope just before F13 in the N-terminal fusions clearly does not interfere with the interaction between PapMV CP monomers that is crucial for self-assembly of nanoparticles.
PapMV NP-12 nanoparticles are better inducers of the CTL response
PapMV NP-12 nanoparticles but not discs are able to trigger a CTL response
In this experiment, our objective was to compare the efficacy of PapMV nanoparticles and discs in triggering a CTL response. PapMV NP-12 discs and nanoparticles were used to immunize (3 immunizations) mice (5 per group) with 100 μg of protein. We measured the production of IFN-μ after stimulation of splenocytes harvested from immunized mice 2 weeks after the second boost using the NP147-155 peptide. The level of IFN-γ secreted by splenocytes specific to PapMV NP-12 nanoparticles was significantly higher than that specific to PapMV NP-12 discs [Figure 2], suggesting that assembly into a highly ordered structure, i.e. nanoparticles, is critical to triggering the CTL response efficiently. We also noted that NP-12 discs appeared less stable than nanoparticles, and initiated aggregation at 34°C as compared to 37°C for nanoparticles [Figure 3]. Discs have the same diameter but are shorter than nanoparticles (30 nm vs 90 nm) and also less stable. These differences in size and stability could account for the observed differences in immunogenicity. Another difference between discs and nanoparticles is the RNA that they contain. Discs are associated with only very small amounts of RNA but nanoparticles contains ssRNA of bacterial origin . It is possible that the ssRNA found in nanoparticles plays a role in the efficacy of the measured immune response. It is known that ssRNA of bacterial origin, as found in PapMV nanoparticles, can be recognized as pathogen associated molecular patterns (PAMPs) by several nucleic acid sensors like RIG-I, MDA-5, TLR7 or TLR8 that are at the interface between the innate and the adaptive immune response [15–17]. We are currently investigating if these sensors play a role in the CTL response.
Our results are consistent with findings obtained with peptide fusions made at the N-terminus of the CP of potato virus X (PVX)—another member of the potexvirus family. It was shown that production of recombinant PVX virus particles in planta can elicit either an humoral  or a CTL immune response . The use of the N-terminus for fusion of peptides on this type of vaccine platform can, however, be problematic if the recombinant virus particles are produced in planta because the fusion may interfere with long-distance transport of the virus throughout the plants and thus affect yield . This is one of the main reasons why we chose to produce our nanoparticles in a bacterial expression system that does not depend on the replication or cell-to-cell transport of the virus.
It is well accepted that the N-terminus of PapMV and potexvirus CP is exposed at the surface of the virus particle [2, 8, 21, 22]. The recently published 3D structure of PapMV CP revealed that the N-terminus is involved in the interaction between two CP subunits in the virus particle , and that the 12 N-terminal residues upstream of F13 are directly exposed on the surface . The availability of the CTL epitope located at this position, as compared to the C-terminus, probably facilitates its cleavage by host proteases and favours loading of the MHC class I pocket in the immune cells [6, 9].
The results of this study improve our understanding of the PapMV vaccine platform and highlight the importance of nanoparticle stability in triggering a CTL response. We can now beneficiate of two different points of fusion for a CTL epitope on the PapMV CP. The fusion at the N-terminus was clearly superior for the NP147-155 peptide but it does not mean that this will be the case for another CTL epitope. The amino acid sequence of the CTL epitope and its influence on the structure on the PapMV CP can have a major impact on its stability and their immunogenicity. Those results are increasing the versatility of the vaccine platform and provide more options for production of stable constructs. Because it is well established that the trigger of a CTL response to conserved epitopes is a valuable approach in the design of prophylactic or therapeutic vaccines to chronic diseases [23–27], we believe that the PapMV vaccine platform will be a very useful tool.
All the work with animals adhered to the Institution-approved ethics protocol of the “Comité de Protection des Animaux” – CHUQ (CPA-CHUQ). The approval of this project is listed under the authorization number 2010148–1.
Cloning and production of PapMV NP constructs
Forward and reverse oligonucleotides used to produce PapMV CP recombinant proteins
Dynamic light scattering
The size of nanoparticles and discs was determined using a ZetaSizer Nano ZS (Malvern, Worcestershire, United Kingdom) at a temperature of 4°C and at a concentration of 0.1 mg/ml in PBS 1x for nanoparticles and at a concentration of 0.25 mg/ml in Tris–HCl 10 mM for discs. The thermal stability of PapMV nanoparticles was measured under the same experimental conditions at temperatures from 24°C to 40°C.
Chemical cross-linking with glutaraldehyde
Cross-linking was performed using 0.1% glutaraldehyde in 10 mM Tris, 50 mM NaCl pH 7.5 in a final volume of 50 μl. The optimal concentration of protein for cross-linking was 150 ng/ml. After addition of glutaraldehyde, the mixture was incubated at room temperature for 30 minutes in the dark. The reaction was stopped with 15 μl of loading dye and heated for 10 minutes at 95°C before separating the proteins by 8% SDS-PAGE. The cross-linked proteins used to immunize mice were stored at 4°C until immunization without adding loading dye.
SDS-PAGE and trypsin digest
Prior to SDS-PAGE, samples were mixed with one-third of the final volume of loading buffer containing 5% SDS, 30% glycerol and 0.01% bromophenol blue and heated for 10 minutes at 95°C. For the trypsin digest, we incubated 10 μg of proteins at 37°C in a volume of 50 μl for 120 minutes in 100 mM Tris–HCl pH 8.5 with 0.2 μg trypsin (Roche, 1418475). The reaction was stopped by adding 10 μl of loading dye. Samples were heated for 10 minutes at 95°C prior to loading on SDS-PAGE .
Five 6- to 8-week-old Balb/C mice (Charles River, Wilmington, MA) were immunized by the intraperitoneal route with: (i) 100 μg of PapMV NP-12 nanoparticles; (ii) 100 μg of PapMV NP-C nanoparticles; (iii) 100 μg of PapMV CP nanoparticles; (iv) 100 μg of PapMV NP-12 discs, and (v) 100 μg of PapMV NP-C discs. Primary immunization was followed by two booster doses given at 2-week intervals. Blood samples were obtained before each injection and 2 weeks after the last one and stored at -20°C until analysis.
Two weeks after the last boost, mice were sacrificed and spleens were recovered for ELISPOT assay performed as described previously . The precursor frequency of specific T cells was determined by subtracting the background spots in media alone from the number of spots seen in wells reactivated with NP147-155 peptide. Data were analyzed with a parametric or a non-parametric ANOVA test when the variances differed significantly and with a Tukey or a Dunn post-test to compare difference among groups of mice. Values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant. Statistical analyses were done with GraphPad PRISM 5.01.
This research project was funded by the Canadian Institute of Health Research Canada (CIHR) (grant number: 185160). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also thank Dr Helen Rothnie for English editing of the manuscript.
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