Nanoparticles Carrying Conserved Regions of Influenza A Hemagglutinin, Nucleoprotein, and M2 Protein Elicit a Strong Humoral and T Cell Immune Response and Protect Animals from Infection.

Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15-35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection.

The extracellular domain of the M2 protein (M2e) and conserved region of the second subunit of the hemagglutinin (HA2) could be used for the development of broad-spectrum vaccines against influenza A. Here we obtained and characterized recombinant mosaic proteins containing tandem copies of M2e and HA2 fused to an artificial self-assembling peptide (SAP). The inclusion of SAP peptides in the fusion proteins enabled their self-assembly in vitro into spherical particles with a size of 30-50 nm. Intranasal immunization of mice with these particles without additional adjuvants induced strong humoral immune response against M2e and the whole virus. Particles carrying both M2e and HA2 induced antigen-specific multifunctional CD4+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with different subtypes of influenza A virus. The obtained self-assembling nanoparticles can be used to develop a universal influenza vaccine.

Highly Immunogenic Nanoparticles Based on a Fusion Protein Comprising the M2e of Influenza A Virus and a Lipopeptide.

The highly conserved extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is a promising target for the development of broad-spectrum vaccines. However, M2e is a poor immunogen by itself and must be linked to an appropriate carrier to induce an efficient immune response. In this study, we obtained recombinant mosaic proteins containing tandem copies of M2e fused to a lipopeptide from Neisseria meningitidis surface lipoprotein Ag473 and alpha-helical linkers and analyzed their immunogenicity. Six fusion proteins, comprising four or eight tandem copies of M2e flanked by alpha-helical linkers, lipopeptides, or a combination of both of these elements, were produced in Escherichia coli. The proteins, containing both alpha-helical linkers and lipopeptides at each side of M2e repeats, formed nanosized particles, but no particulate structures were observed in the absence of lipopeptides. Animal study results showed that proteins with lipopeptides induced strong M2e-specific antibody responses in the absence of external adjuvants compared to similar proteins without lipopeptides. Thus, the recombinant M2e-based proteins containing alpha-helical linkers and N. meningitidis lipopeptide sequences at the N- and C-termini of four or eight tandem copies of M2e peptide are promising vaccine candidates.

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus.

Hepatitis C virus (HCV) can cause chronic infection and evade the immune response. The generation and maintenance of an effective T-cell response is important for immune-mediated control of HCV infection. The purpose of this study was to obtain recombinant mosaic proteins containing the cytotoxic T lymphocyte (CTL) epitopes of HCV fused with different adjuvants and analyse their immunogenicity. A recombinant polyepitope protein comprising HLA-A2-restricted CTL epitopes of the NS3, NS4ab and NS5a proteins of HCV was designed. Adjuvant compounds, the T-helper (Th) epitope PADRE, lipopeptide from Neisseria meningiditis and interleukin 2 (IL-2) were included in the fusion proteins. Three proteins differing in their adjuvant content were expressed in Escherichia coli and purified. The purified proteins formed nanosized particles. The proteins were characterized by their ability to cause proliferation of spleen cells, induce expression of cytokine genes and production of interferon gamma by T lymphocytes of immunized mice. The obtained recombinant vaccine proteins effectively stimulate dendritic cells, which in turn specifically activate Th1 and Th2 lymphocytes. Adjuvant components act additively to enhance the stimulation of dendritic cells and polarize them in the direction of Th1 lymphocyte activation. Analysis of spleen cell proliferation, expression of Th1 and Th2 cytokines and production of interferon gamma by lymphocytes of immunized mice after specific stimulation in vitro revealed that recombinant protein comprising CTL epitopes of HCV, Th epitope PADRE, lipoprotein and IL-2 induced the highest response of T-lymphocytes.

High immunogenicity of plant-produced candidate influenza vaccine based on the M2e peptide fused to flagellin.

The ectodomain of the conserved influenza matrix protein M2 (M2e) is a promising target for the development of a universal influenza vaccines. Immunogenicity of M2e could be enhanced by its fusion to bacterial flagellin, the ligand for Toll-like receptor 5. Previously we reported the transient expression in plants of a recombinant protein Flg-4M comprising flagellin fused to 4 tandem copies of the M2e. The use of self-replicating recombinant vector based on the potato virus X allowed expression of Flg-4M in Nicotiana benthaminana leaves at a very high level, up to about 1 mg/g of fresh leaf tissue. Intranasal immunization of mice with Flg-4M induced M2e-specific serum antibodies and provided protection against lethal challenge with different strains of influenza A virus. Here we show that immunization with Flg-4M not only generates a strong immune response, but also redirects the response from the carrier flagellin toward the M2e epitopes. Significant IgG response to M2e was also developed in bronchoalveolar lavages of immunized mice. Protective activity of Flg-4M upon lethal influenza challenge correlated with a decrease of virus titers in lungs relative to the control. Overall these data show the potential for the development of a plant-produced M2e-flagellin universal influenza vaccine.

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant loop region of hepatitis B core antigen: Insertion of multiple copies of M2e increases immunogenicity and protective efficiency.

The extracellular domain of the transmembrane protein M2 (M2e) of influenza A virus is a promising target for the development of "universal" vaccines against influenza. M2e is a poor immunogen by itself; however, when M2e is linked to an appropriate carrier, such as hepatitis B virus core (HBc) particles, it becomes highly immunogenic. Insertions of target peptides into the surface-exposed major immunodominant loop region (MIR) of the HBc antigen are especially immunogenic, but such insertions often affect the protein folding and formation of recombinant virus-like particles. To facilitate an appropriate conformation of the M2e insert, we introduced flexible linkers at the junction points between the insert and flanking HBc sequences. This approach allowed the construction of recombinant HBc particles carrying 1, 2 and 4 copies of M2e in the MIR region. These particles were produced in Escherichia coli and purified to homogeneity. The immune response and protective activity of hybrid HBc particles in mice correlated with the number of inserted M2e peptides: the highest immunogenicity and complete protection of mice against the lethal challenge by influenza virus was observed with particles carrying four copies of M2e. The possibility of the simultaneous presentation of M2e peptides from several important influenza strains on a single HBc particle could also facilitate the development of a broad-specificity vaccine efficient not only against influenza A strains of human origin but also for newly emerging strains of animal origin, such as the avian influenza.

Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors.

BACKGROUND: The extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e. RESULTS: In this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30% of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. CONCLUSIONS: This study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant region of hepatitis B core antigen: Broad protective efficacy of particles carrying four copies of M2e.

A long-term objective when designing influenza vaccines is to create one with broad cross-reactivity that will provide effective control over influenza, no matter which strain has caused the disease. Here we summarize the results from an investigation into the immunogenic and protective capacities inherent in variations of a recombinant protein, HBc/4M2e. This protein contains four copies of the ectodomain from the influenza virus protein M2 (M2e) fused within the immunodominant loop of the hepatitis B virus core antigen (HBc). Variations of this basic design include preparations containing M2e from the consensus human influenza virus; the M2e from the highly pathogenic avian A/H5N1 virus and a combination of two copies from human and two copies from avian influenza viruses. Intramuscular delivery in mice with preparations containing four identical copies of M2e induced high IgG titers in blood sera and bronchoalveolar lavages. It also provoked the formation of memory T-cells and antibodies were retained in the blood sera for a significant period of time post immunization. Furthermore, these preparations prevented the death of 75-100% of animals, which were challenged with lethal doses of virus. This resulted in a 1.2-3.5 log10 decrease in viral replication within the lungs. Moreover, HBc particles carrying only "human" or "avian" M2e displayed cross-reactivity in relation to human (A/H1N1, A/H2N2 and A/H3N2) or A/H5N1 and A(H1N1)pdm09 viruses, respectively; however, with the particles carrying both "human" and "avian" M2e this effect was much weaker, especially in relation to influenza virus A/H5N1. It is apparent from this work that to quickly produce vaccine for a pandemic it would be necessary to have several variations of a recombinant protein, containing four copies of M2e (each one against a group of likely influenza virus strains) with these relevant constructs housed within a comprehensive collection Escherichia coli-producers and maintained ready for use.

Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.

A molecular assembly system for presentation of antigens on the surface of HBc virus-like particles.

Hepatitis B virus-like particles, icosahedral structures formed by multiple core protein dimers, are promising immune-enhancing vaccine carriers for foreign antigens. Insertions into the surface-exposed immunodominant loop are especially immunogenic. However, the need to conserve the particulate structure to ensure high immunogenicity imposes restraints on the nature of the heterologous sequence that can be inserted. We propose a new approach to constructing HBc particles linked to the target epitopes that relies on non-covalent interactions between the epitope and pre-assembled unmodified HBc particles. Interaction was enabled by fusion of the epitope to the GSLLGRMKGA peptide, binding to the spike tips. This peptide may be used as a "binding tag" allowing in vitro construction of HBc particles carrying the target peptide. Such virus-like particles carrying multiple copies of the extracellular domain of the M2 protein of different influenza strains appeared to be highly immunogenic and protected immunised mice against a lethal influenza challenge.

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage.

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.