M2e nanovaccines supplemented with recombinant hemagglutinin protect chickens against heterologous HPAI H5N1 challenge.

Current poultry vaccines against influenza A viruses target the globular head region of the hemagglutinin (HA1), providing limited protection against antigenically divergent strains. Experimental subunit vaccines based on the conserved ectodomain of the matrix protein 2 (M2e) induce cross-reactive antibody responses, but fail to fully prevent virus shedding after low pathogenic avian influenza (LPAI) virus challenge, and are ineffective against highly pathogenic avian influenza (HPAI) viruses. This study assessed the benefits of combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine in protecting chickens against a heterologous HPAI H5N1 virus challenge.

Intranasal delivery of a self-adjuvanted nanovaccine composed of the curli filaments and the highly conserved M2e epitope confers protection against influenza a virus in mice.

Intranasal administration of vaccines is an attractive delivery route to fight viral respiratory infections. However, there are only a few intranasal vaccines used in human, emphasizing the critical need to identify novel safe mucosal adjuvants and antigen delivery systems to expand their usage. We recently revealed an immunostimulating nanoparticle based on a fragment (R4R5) of the Curli-specific gene A (CsgA) protein that confers protection against influenza A virus (IAV) when conjugated to three repeats of the highly conserved M2e epitope and administrated intramuscularly.

Bionanocomposites with Enhanced Physical Properties from Curli Amyloid Assemblies and Cellulose Nanofibrils.

Proteinaceous amyloid fibrils are one of the stiffest biopolymers due to their extensive cross-β-sheet quaternary structure, whereas cellulose nanofibrils (CNFs) exhibit interesting properties associated with their nanoscale size, morphology, large surface area, and biodegradability. Herein, CNFs were supplemented with amyloid fibrils assembled from the Curli-specific gene A (CsgA) protein, the main component of bacterial biofilms. The resulting composites showed superior mechanical properties, up to a 7-fold increase compared to unmodified CNF films.

Synthetic Multicomponent Nanovaccines Based on the Molecular Co-assembly of β-Peptides Protect against Influenza A Virus.

Peptides with the ability to self-assemble into nanoparticles have emerged as an attractive strategy to design antigen delivery platforms for subunit vaccines. While toll-like receptor (TLR) agonists are promising immunostimulants, their use as soluble agents is limited by their rapid clearance and off-target inflammation. Herein, we harnessed molecular co-assembly to prepare multicomponent cross-β-sheet peptide nanofilaments exposing an antigenic epitope derived from the influenza A virus and a TLR agonist.

Engineered Curli Nanofilaments as a Self-Adjuvanted Antigen Delivery Platform.

Proteinaceous nanoparticles constitute efficient antigen delivery systems in vaccine formulations due to their size and repetitive nature that mimic most invading pathogens and promote immune activation. Nonetheless, the coadministration of an adjuvant with subunit nanovaccines is usually required to induce a robust, long-lasting, and protective immune response. Herein, the protein Curli-specific gene A (CsgA), which is known to self-assemble into nanofilaments contributing to bacterial biofilm, is exploited to engineer an intrinsically immunostimulatory antigen delivery platform.

Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds.

Vaccination has saved billions of human lives and has considerably reduced the economic burden associated with pandemic and endemic infectious diseases. Notwithstanding major advancements in recent decades, multitude diseases remain with no available effective vaccine. While subunit-based vaccines have shown great potential to address the safety concerns of live-attenuated vaccines, their limited immunogenicity remains a major drawback that still needs to be addressed for their use fighting infectious illnesses, autoimmune disorders, and/or cancer.

Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5).

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist.

Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB.

Flagellin constitutes a potential adjuvant for vaccines owing to its robust immunostimulatory properties. However, clinical trials have revealed that flagellin derived from Salmonella enterica serovar Typhimurium induces high levels of proinflammatory markers and substantial adverse effects. The flagellin from Bacillus subtilis, Hag, shares high sequence homology with Salmonella FljB within the D0 and D1 domains responsible for TLR5 engagement, while the D2 and D3 domains associated with an off-target immune response are absent.

Self-assembled peptide nanorod vaccine confers protection against influenza A virus.

Proteinaceous nanostructures have emerged as a promising strategy to develop safe and efficient subunit vaccines. The ability of synthetic β-sheet self-assembling peptides to stabilize antigenic determinants and to potentiate the epitope-specific immune responses have highlighted their potential as an immunostimulating platform for antigen delivery. Nonetheless, the intrinsic polymorphism of the resulting cross-β fibrils, their length in the microscale and their close structural similarity with pathological amyloids could limit their usage in vaccinology.

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-β Fibrils to Design Adjuvanted Nanovaccines.

Protein fibrils characterized with a cross-β-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling β-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus.

Identification of the nuclear and nucleolar localization signals of the Feline immunodeficiency virus Rev protein.

Lentivirus genomes code for a regulatory protein essential for virus replication termed Rev. The Rev protein binds to partially spliced and unspliced viral RNAs and mediates their nuclear export. Therefore, Rev possesses functional domains that enable its shuttling between the cytoplasm and the nucleus.

Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein.

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown.

Protein Supramolecular Structures: From Self-Assembly to Nanovaccine Design.

Life-inspired protein supramolecular assemblies have recently attracted considerable attention for the development of next-generation vaccines to fight against infectious diseases, as well as autoimmune diseases and cancer. Protein self-assembly enables atomic scale precision over the final architecture, with a remarkable diversity of structures and functionalities. Self-assembling protein nanovaccines are associated with numerous advantages, including biocompatibility, stability, molecular specificity and multivalency.

The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals.

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus.

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods.

Peptides that self-assemble into cross-β-sheet amyloid structures constitute promising building blocks to construct highly ordered proteinaceous materials and nanoparticles. Nevertheless, the intrinsic polymorphism of amyloids and the difficulty of controlling self-assembly currently limit their usage. In this study, the effect of electrostatic interactions on the supramolecular organization of peptide assemblies is investigated to gain insights into the structural basis of the morphological diversities of amyloids.

Nanoparticle-Based Vaccines Against Respiratory Viruses.

The respiratory mucosa is the primary portal of entry for numerous viruses such as the respiratory syncytial virus, the influenza virus and the parainfluenza virus. These pathogens initially infect the upper respiratory tract and then reach the lower respiratory tract, leading to diseases. Vaccination is an affordable way to control the pathogenicity of viruses and constitutes the strategy of choice to fight against infections, including those leading to pulmonary diseases.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform.

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined.

The Cholera Toxin B Subunit (CTB) Fused to the Porcine Arterivirus Matrix M and GP5 Envelope Proteins Fails to Enhance the GP5-Specific Antibody Response in Pigs Immunized with Adenovectors.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of the Arteriviridae family. As the current commercial vaccines are incompletely protective effective against PRRSV infection, we developed a vaccine strategy using replicating but non-disseminating adenovectors (rAdVs) expressing the PRRSV M matrix protein in fusion with the neutralizing major epitope-carrying GP5 envelope protein (Roques et al. in Vet Res 44:17, 2013).

Flagellin in fusion with human rotavirus structural proteins exerts an adjuvant effect when delivered with replicating but non-disseminating adenovectors through the intrarectal route.

Human rotavirus (HRV) is the worldwide leading cause of gastroenteritis in young children. Two live attenuated HRV vaccines have been approved since 2006. However, these live vaccines still have potential risks including reversion of virulence.

Antibody responses induced in mice immunized with recombinant adenovectors expressing chimeric proteins of various porcine pathogens.

Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine Circovirus type 2 (PCV2) and Mycoplasma (M.) hyopneumoniae are swine pathogens of economic importance. Although vaccines are available for each pathogen, no commercial bivalent vaccines have been developed so far.