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

Optimisation de la réponse cellulaire induite par les nanoparticules du PapMV fusionnées à un épitope CTL du virus Influenza

La protéine de capside du virus de la mosaïque de la papaye (PapMV CP) s’assemble autour d’un ARN simple brin pour former des nanoparticules. Nous avons montré que ces nanoparticules peuvent être fusionnées à des épitopes peptidiques permettant le développement d’une réponse immunitaire protectrice. Le C-terminal de la protéine a été utilisé comme site de fusion dans nos études précédentes. Récemment, nous avons découvert que des fusions après le résidu 12 ou 187 de la CP démontrent aussi un potentiel pour fusionner les épitopes d’intérêt. Le but de ce projet de recherche est de déterminer la capacité d’induire une réponse cellulaire en utilisant des nanoparticules PapMV CP fusionnées à un épitope cellulaire sur différents sites de fusion. L’immunisation de souris avec les particules recombinantes a permis de démontrer qu’une fusion au N-terminus était plus efficace pour induire la prolifération des lymphocytes T CD8+ spécifiques. De plus, les résultats révèlent que la multimérisation de la CP sous forme de nanoparticules est un critère requis pour induire une réponse cellulaire

PapMV amino acid sequence and structure.

<p>PapMV capsid protein amino acid sequence with each site of fusion and a bioinformatic secondary structure prediction (adapted from Lecours 2006).</p

Forward and reverse oligonucleotides used to produce the seven PapMV-HA11 recombinant proteins.

<p>The sequences in bold and ithalic correspond to the <i>Acc651</i> restriction site.</p

Structural changes in PapMV CP in the different recombinant nanoparticles induced by an increase in temperature.

<p>Each of the recombinant nanoparticles (PapMV-HA11-12, PapMV-HA11-187 and PapMV-HA11-C) at a concentration of 0.1 mg/ml were heated in steps of 1°C and secondary structure changes of the protein was monitored by circular dichroism. The read-out was performed at a wave length of 208 nm. The arrows show the point of inflection for each of the nanoparticles. The black bar represents the body temperature of mice (36.9°C).</p

Stable nanoparticles are more immunogenic in animals.

<p>Balb/C mice (5 per groups) were immunized twice with a 14-day interval with 100 µg s.c. of PapMV-HA11-12, PapMV-HA11-187 or PapMV-HA11-C, respectively. The total IgG (A) or the IgG2a (B) humoral response directed to the HA11 peptide was measured by ELISA. Also, the total IgG (C) and IgG2a (D) directed to the PapMV CP was measured by ELISA. *** P<0.0001 **P<0.01.</p

Aggregation of recombinant nanoparticles upon heating.

<p>Each of the recombinant nanoparticles at a concentration of 0.1 mg/ml were heated by steps of 5°C and DLS spectra of the samples was measured. The approximate size of the particles measured indicates the level of aggregation of the samples. The arrows show the point of inflection for each of the nanoparticles. The black bar represents the body temperature of mice (36.9°C).</p

The M3M2 antigen mixed with the OmpC adjuvant induces a strong humoral response to the H3N8 M2e peptide.

<p>Balb/C mice (10 per group) were immunized twice with either 100µg of the recombinant M3M2 antigen or with 23µg of the H3N8 M2e peptide. Total IgG titers (A) or IgG2a (B) directed to the H3N8 M2e peptide were evaluated by ELISA using serum collected 14 days after the second immunization. Using a similar immunization protocol, Balb/C mice were immunized with 20µg of M3M2 alone or mixed with one of several doses of the adjuvant OmpC (OC) (20, 40, 60 or 80µg) to increase the immune response to the M2e peptide. Total IgG (C) and IgG2a (D) were evaluated from serum collected 14 days after the second immunization. Finally, to evaluate if the vaccine formulations could trigger a memory response, ELISA was performed with serum collected at day 61 after the second immunization of the same animals as in C and D. Total IgG (E) and IgG2a (F) directed to the peptide H3N8 M2e were measured. Adjuvanted groups unified under one arc means that they are all significantly different from the non-adjuvanted group with the same level of confidence showed with the above capped line. * p< 0.05, ** p< 0.01, *** p< 0.001.</p

The serum obtained from mice immunized with the optimized M3M2/OC vaccine shows cross reactivity with divergent M2e peptides.

<p>(A) Amino acid sequence of the divergent M2e peptides derived from the avian H5N1, H9N2 or the human H1N1 strain. The serum used to perform ELISA was collected from mice immunized in the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077084#pone-0077084-g002" target="_blank">Figure 2A</a>. Total IgG directed (B) IgG2a to H5N1 M2e, total IgG (C) and IgG2a (D) to H9N2 M2e and total IgG (E) and IgG2a (F) to the H1N1 M2e. * p< 0.05 and *** p< 0.001.</p