Plant-virus-based vaccines have emerged as a promising avenue in vaccine development. This report describes the engineering of an innovative vaccine platform using the papaya mosaic virus (PapMV) capsid protein (CP) as a carrier protein and a C-terminal fused hepatitis C virus (HCV) E2 epitope as the immunogenic target. Two antigen organizations of the PapMV-based vaccines were tested: a virus-like-particle (VLP; PapMVCP-E2) and a monomeric form (PapMVCP(27-215)-E2). While the two forms of the vaccine were both shown to be actively internalized in vitro in bone-marrow-derived antigen presenting cells (APCs), immunogenicity was demonstrated to be strongly dependent on antigen organization. Indeed, C3H/HeJ mice injected twice with the multimeric VLP vaccine showed a long-lasting humoral response (more than 120 days) against both the CP and the fused HCV E2 epitope. The antibody profile (production of IgG1, IgG2a, IgG2b, IgG3) suggests a Th1/Th2 response. Immunogenicity of the PapMV vaccine platform was not observed when the monomer PapMVCP-E2 was injected. These results demonstrate for the first time the potential of the PapMV vaccine platform and the critical function of multimerization in its immunogenicity.

Papaya mosaic potexvirus (PapMV) is a member of the potexvirus family. The virion is a flexible rod 500 nm in length and 15 nm in diameter composed of 1400 subunits of viral coat protein (CP) [1] assembled around a plus-strand genomic RNA of 6656 nucleotides (nt) [2]. In vitro reconstitution of PapMV nucleocapsid-like particles (NLPs) was previously studied using CP prepared from the acetic acid degradation of purified virus [3]. Using this method, PapMV CP can be isolated from the genomic RNA and used for in vitro assembly assays [3]. Extracted CP has been found as a variety of aggregates ranging from 14S to 25S [4] that include a disk-like structure (14 S) made of 18-20 subunits. These disks are helical structures (two turns of the helix) similar in architecture to the native virus particle [3,5]. The addition of RNA to the isolated disks triggers the assembly of long rod-shaped particles that are very similar to the WT virus [3]. The in vitro assembly of PapMV was shown to be specific and triggered by 47 nucleotides of the 5¢ noncoding region of the virus [6]. This region is free of any discernable secondary structure, an important feature for initiation of the assembly process [6]. In vitro assembly is specific when performed at a pH of 8.0-8.5 in a buffer of low ionic strength [4] and elongation proceeds in the 5¢fi3¢ direction [7]. One atom of Ca 2+ is attached to each subunit, which is probably important for the structure of the protein [8]. Alignment of PapMV CP with the CP of other potexviruses reveals that these proteins share 35% identity [9]. The N terminus of the proteins is the most divergent and their length is also variable. It can reach more than 50 amino acids in the case of potato aucuba mosaic virus. Phosphorylation of the N terminus of potato virus X CP by host Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPDN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPDN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPDN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.Abbreviations CP, coat protein; NLP, nucleocapsid like particles; nt, nucleotide; PapMV, Papaya mosaic potexvirus.

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

Using a polyclonal antibody generated against the purified pea (Pisum sativum) carbonic anhydrase (CA) monomeric species, we have isolated and characterized a cDNA coding for this enzyme. Protein sequence analysis was used to confirm the identity of the clone. The presence of a large transit peptide suggests that CA is transported into the chloroplast and then processed to the mature size of approximately 26 kilodaltons. Northern hybridization, using the CA cDNA as a probe of total leaf RNA, revealed a single transcript of 1.45 kilobase pairs. This transcript was not detected in RNA extracted from root or etiolated leaf tissue. Comparison of the deduced amino acid sequence with that of spinach CA showed approximately 68% identity over the length of the nascent protein but with greater similarity observed within the mature protein sequences. In addition, regions of the pea and spinach CA proteins were found to be significantly similar to the Escherichia coli cyanate permease.One of the more abundant soluble proteins in the leaves of higher plants is the

Commercial seasonal flu vaccines induce production of antibodies directed mostly towards hemaglutinin (HA). Because HA changes rapidly in the circulating virus, the protection remains partial. Several conserved viral proteins, e.g., nucleocapsid (NP) and matrix proteins (M1), are present in the vaccine, but are not immunogenic. To improve the protection provided by these vaccines, we used nanoparticles made of the coat protein of a plant virus (papaya mosaic virus; PapMV) as an adjuvant. Immunization of mice and ferrets with the adjuvanted formulation increased the magnitude and breadth of the humoral response to NP and to highly conserved regions of HA. They also triggered a cellular mediated immune response to NP and M1, and long-lasting protection in animals challenged with a heterosubtypic influenza strain (WSN/33). Thus, seasonal flu vaccine adjuvanted with PapMV nanoparticles can induce universal protection to influenza, which is a major advancement when facing a pandemic.