A nanoparticle-based COVID-19 vaccine candidate elicits broad neutralizing antibodies and protects against SARS-CoV-2 infection.

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity.

BACKGROUND: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. METHODS: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. CONCLUSIONS: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

A Randomized Controlled Study to Evaluate the Safety and Reactogenicity of a Novel rVLP-Based Plant Virus Nanoparticle Adjuvant Combined with Seasonal Trivalent Influenza Vaccine Following Single Immunization in Healthy Adults 18-50 Years of Age.

Inactivated influenza vaccines efficacy is variable and often poor. We conducted a phase 1 trial (NCT02188810), to assess the safety and immunogenicity of a novel nanoparticle Toll-like receptor 7/8 agonist adjuvant (Papaya Mosaic Virus) at different dose levels combined with trivalent influenza vaccine in healthy persons 18-50 years of age. Hemagglutination-inhibition assays, antibody to Influenza A virus nucleoprotein and peripheral blood mononuclear cells for measurement of interferon-gamma ELISPOT response to influenza antigens, Granzyme B and IFNγ:IL-10 ratio were measured. The most common adverse events were transient mild to severe injection site pain and no safety signals were observed. A dose-related adjuvant effect was observed. Geometric mean hemagglutination-inhibition titers increased at day 28 in most groups and waned over time, but fold-antibody responses were poor in all groups. Cell mediated immunity results were consistent with humoral responses. The Papaya Mosaic Virus adjuvant in doses of 30 to 240 µg combined with reduced influenza antigen content was safe with no signals up to 3 years after vaccination. A dose-related adjuvant effect was observed and immunogenicity results suggest that efficacy study should be conducted in influenza antigen-naïve participants.

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.

The quest for a nanoparticle-based vaccine inducing broad protection to influenza viruses.

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.

Activation of innate immunity in primary human cells using a plant virus derived nanoparticle TLR7/8 agonist.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

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.

Influence of PapMV nanoparticles on the kinetics of the antibody response to flu vaccine.

BACKGROUND: The addition of an adjuvant to a vaccine is a promising approach to increasing strength and immunogenicity towards antigens. Despite the fact that adjuvants have been used in vaccines for decades, their mechanisms of action and their influence on the kinetics of the immune response are still not very well understood. The use of papaya mosaic virus (PapMV) nanoparticles-a novel TLR7 agonist-was recently shown to improve and broaden the immune response directed to trivalent inactivated flu vaccine (TIV) in mice and ferrets. RESULTS: We investigated the capacity of PapMV nanoparticles to increase the speed of the immune response toward TIV. PapMV nanoparticles induced a faster and stronger humoral response to TIV that was measured as early as 5 days post-immunization. The addition of PapMV nanoparticles was shown to speed up the differentiation of B-cells into early plasma cells, and increased the growth of germinal centers in a CD4+ dependent manner. TIV vaccination with PapMV nanoparticles as an adjuvant protected mice against a lethal infection as early as 10 days post-immunization. CONCLUSION: In conclusion, PapMV nanoparticles are able to accelerate a broad humoral response to TIV. This property is of the utmost importance in the field of vaccination, especially in the case of pandemics, where populations need to be protected as soon as possible after vaccination.

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.

PapMV nanoparticles improve mucosal immune responses to the trivalent inactivated flu vaccine.

BACKGROUND: Trivalent inactivated flu vaccines (TIV) are currently the best means to prevent influenza infections. However, the protection provided by TIV is partial (about 50%) and it is needed to improve the efficacy of protection. Since the respiratory tract is the main site of influenza replications, a vaccine that triggers mucosal immunity in this region can potentially improve protection against this disease. Recently, PapMV nanoparticles used as an adjuvant in a formulation with TIV administered by the subcutaneous route have shown improving the immune response directed to the TIV and protection against an influenza challenge. FINDINGS: In the present study, we showed that intranasal instillation with a formulation containing TIV and PapMV nanoparticles significantly increase the amount of IgG, IgG2a and IgA in lungs of vaccinated mice as compared to mice that received TIV only. Instillation with the adjuvanted formulation leads to a more robust protection against an influenza infection with a strain that is lethal to mice vaccinated with the TIV. CONCLUSIONS: We demonstrate for the first time that PapMV nanoparticles are an effective and potent mucosal adjuvant for vaccination.

Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus.

Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.

Improvement of the PapMV nanoparticle adjuvant property through an increased of its avidity for the antigen [influenza NP].

The principal caveat of existing influenza vaccine is their failure to provide long-term protection. This lack of efficiency is caused by persistent (drift) and dramatic (shift) antigenic changes on the major surface proteins, the main target of protective immunity generated by traditional vaccines. Alternatively, vaccination with most conserved protein, like the nucleoprotein (NP) can stimulate immunity against multiple serotypes and could potentially provides an extended protection. The NP antigen contains more than 90% protein sequence homology among influenza A isolates and it also contains dominant CTL targets epitopes that made this antigen an attractive target for developing universal vaccine. However, NP protein is a weak antigen and need the use of adjuvant to increase its immunogenicity. We have developed an innovative high avidity VLP (HAV) nanoparticle to improve its adjuvant property to the NP antigen. The nanoparticles are derived from papaya mosaic virus capsid protein (PapMV CP) produced in a bacteria expression system. We generated the HAV by adding an affinity peptide directed to the NP protein at the surface of the VLPs. The fusions of the affinity peptide to PapMV VLPs increased the avidity of PapMV VLPs to NP protein. This modification enhanced the humoral and the IFN-γ response directed to NP. Moreover, the immunity generated by the HAV adjuvanted NP vaccine improved the protection of vaccinated mice to a challenge with influenza virus. The protection was characterized by accelerated virus elimination after the onset of infection and rapid recovery of the vaccinated animals.

Improvement of the trivalent inactivated flu vaccine using PapMV nanoparticles.

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.

Two distinct chimeric potexviruses share antigenic cross-presentation properties of MHC class I epitopes.

Chimeric VLPs made of papaya mosaic virus (PapMV) trigger a CTL response through antigenic presentation of epitopes on MHC class I. Here, a chimeric VLP composed of malva mosaic virus (MaMV) was shown to share similar properties. We demonstrated the capacity of both VLPs to enter human APCs. The chimeric constructions were cross-presented in CD40-activated B lymphocytes leading to in vitro expansion of antigen-specific T lymphocytes. We showed that high concentrations of chimeric MaMV induced cell death, suggesting that some modifications can trigger collateral effects in vitro. Results suggest that potexvirus VLPs are an attractive vaccine platform for inducing a CTL response.

Nucleotide sequence and phylogenetic analysis of a new potexvirus: Malva mosaic virus.

A filamentous virus isolated from Malva neglecta Wallr. (common mallow) and propagated in Chenopodium quinoa was grown, cloned and the complete nucleotide sequence was determined (GenBank accession # DQ660333). The genomic RNA is 6858 nt in length and contains five major open reading frames (ORFs). The genomic organization is similar to members and the viral encoded proteins shared homology with the group of the Potexvirus genus in the Flexiviridae family. Phylogenetic analysis revealed a close relationship with narcissus mosaic virus (NMV), scallion virus X (ScaVX) and, to a lesser extent, to Alstroemeria virus X (AlsVX) and pepino mosaic virus (PepMV). A novel putative pseudoknot structure is predicted in the 3'-UTR of a subgroup of potexviruses, including this newly described virus. The consensus GAAAA sequence is detected at the 5'-end of the genomic RNA and experimental data strongly suggest that this motif could be a distinctive hallmark of this genus. The name Malva mosaic virus is proposed.

Effect of cAMP-dependent protein kinase A (PKA) on HCV nucleocapsid assembly and degradation.

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.

Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization.

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.

Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein.

Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) 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 CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.

Signal peptide peptidase promotes the formation of hepatitis C virus non-enveloped particles and is captured on the viral membrane during assembly.

The maturation of the core protein (C) of Hepatitis C virus (HCV) is controlled by the signal peptidase (sp) and signal peptide peptidase (spp) of the host. To date, it remains unknown whether spp cleavage influences viral infectivity and/or the assembly process. Here, evidence is provided that cleavage by spp is not required for assembly of nucleocapsid-like particles (NLPs) in yeast (Pichia pastoris). The immature NLPs (not processed by spp) show a density of 1.11 g ml(-1) on sucrose gradients and a diameter of 50 nm. Co-expression of human spp (hspp) with C generates the 21 kDa mature form of the protein and promotes the accumulation of non-enveloped particles. The amount of non-enveloped particles accumulating in the cell was correlated directly with the expression level of hspp. Furthermore, immunocapture studies showed that hspp was embedded in the membranes of enveloped particles. These results suggest that maturation of the C protein can occur after formation of the enveloped particles and that the abundance of hspp influences the types of particle accumulating in the cells.

The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation.

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.