Influenza Matrix Protein Research Articles

The Development of a Novel Broad-Spectrum Influenza Polypeptide Vaccine Based on Multi-Epitope Tandem Sequences

Background: Polypeptide vaccines have the potential to improve immune responses by targeting conserved and weakly immunogenic regions in antigens. This study aimed to identify and evaluate the efficacy of a novel influenza universal vaccine candidate consisting of multiple polypeptides derived from highly conserved regions of influenza virus proteins hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Methods: Immunoinformatics tools were used to screen conserved epitopes from different influenza virus subtypes (H1N1, H3N2, H5N1, H7N9, H9N2, and IBV). A polypeptide vaccine, P125-H, was constructed by linking multiple epitopes using Ii-Key technology. The immunogenicity of P125-H was assessed in mice using MF59-adjuvanted P125-H via intraperitoneal injection. Hemagglutination inhibition (HI) and neutralizing antibody responses were measured, along with IFN-γ levels in spleen lymphocytes. Protective efficacy was evaluated using viral challenge with lethal doses of H1N1 and H7N9. Results: Mice immunized with P125-H generated high levels of HI and neutralizing antibodies against multiple influenza strains. IFN-γ production was significantly elevated in spleen lymphocytes upon stimulation with the vaccine. P125-H protected mice from influenza infection, reducing weight loss and the viral load in the lungs, mitigating lung pathology, and decreasing mortality. Conclusions: The P125-H vaccine induced broad cross-protection against multiple influenza strains and elicited robust immune responses. It demonstrates strong potential as a candidate for a universal influenza vaccine.

M2e-specific antibodies protect against influenza PR8 virus in an isotype and route dependent manner.

The ectodomain of influenza matrix protein 2 (M2e) is a promising target for the development of universal prophylactic and therapeutic agents against influenza viruses of different subtypes. We constructed three M2e-specific monoclonal antibody variants, M2A1-1 (IgG1), M2A1-2a (IgG2a), M2A1-2b (IgG2b), which have the same Fab region targeting the M2e epitope but different isotypes, and compared their protective efficacy in influenza PR8-infected mice. We found that anti-M2e antibodies provided protection against influenza virus in a subtype-dependent manner, with the IgG2a variant providing significantly better protection with lower virus titers and milder lung injury than IgG1 and IgG2b isotypes. Additionally, we observed that the protective efficacy was dependent on the administration routes, with intranasal administration of antibody providing better protection than intraperitoneal administration. The timing of administration was also critical in determining the protective efficacy; while all the antibody isotypes provided protection when administered before influenza challenge, only IgG2a provided minimal protection when the antibodies were administered after virus challenge. These results provide valuable information for optimizing the therapeutics usage of M2e-based antibodies and furthering the development of M2e-based universal influenza vaccines.

2318. A Composite Peptide Vaccine Comprised of Conserved SARS-CoV-2 and Influenza Epitopes Generated Antisera Reponses to Both Coronavirus and Influenza

Abstract Background While influenza and SARS-CoV-2 virus can be severe, co-infection with these viruses has the potential for worsening the clinical outcome. The combined infection of influenza A virus (IAV) and SARS-CoV-2 in animal models has also shown elevated SARS-CoV-2 viral load and pneumonia. Co-vaccination may provide a useful option in prevention of combined infection. In this study, a composite peptide vaccine comprised of highly conserved influenza neuraminidase (NA) and matrix protein (M2e) epitopes, in combination with either conserved spike protein (SP) or RNA polymerase (POL) epitopes of SARS-CoV-2 generated robust immune responses to both coronavirus and influenza virus. Methods Mice were immunized with 20µg of Coronavirus Pep02 (CorPep02, comprised of POL) or Coronavirus Pep05 (CorPep05, comprised of POL and NA+M2e) or Coronavirus Pep11 (CorPep11, comprised of SP and NA+M2e), formulated with AddaVax™. Serum antibody titers to both coronavirus and influenza peptides, and whole viruses were analyzed using ELISA. Antisera neutralizing activity against IAV was determined using microneutralization assay (MNA). Results CorPep05 immunization generated enhanced IgG1 and IgG2b antisera responses to the immunogens compared to CorPep02, starting at Day-21. Immunization with CorPep05 or CorPep11 induced strong IgG1 and IgG2b immune responses to the composite coronavirus peptides, influenza NA and M2e peptides, coronavirus POL and SP peptides. Antisera from CorPep05 and CorPep11 groups bound to both viruses (IAV and human coronavirus NL-63 (HCoV NL-63)) and demonstrated neutralizing activity against IAV, with titers >5000. Conclusion A composite peptide vaccine comprised of both influenza (NA+M2e) and coronavirus (POL) epitopes generated enhanced immune responses in comparison to peptide containing only POL. Th1 and Th2 immune responses to coronavirus POL and SP were observed. Serum neutralizing activity was demonstrated against IAV. Studies are underway to examine neutralizing antibodies against HCoV NL-63 and multiple variants of SARS-CoV-2. Highly conserved epitopes in these composite peptide vaccines may provide an important strategy to prevent infections with IAV or SARS-CoV-2 and mitigate the threat of co-infection. Disclosures All Authors: No reported disclosures.

RM2e-ΔPly protein immunization induces protection against influenza viruses and its co-infection with Streptococcus pneumoniae in mice

Current seasonal influenza A virus (IAV) vaccines only protect against specific virus and require annual reconstitution to accommodate the viral mutations. A universal influenza vaccination that protects against all IAV strains is urgently needed. The influenza matrix protein 2 ectodomain (M2e) is a potential universal IAV vaccine candidate, but it has a low immunogenicity. ΔA146Ply was proved to be an effective protein adjuvant. Therefore, ΔA146Ply was used as an adjuvant of M2e in this study to evaluate its protective effect against IAV-related infection. Herein, a novel rM2e protein containing multiple M2e originated from different species of IAV was constructed and expressed in Escherichia coli (E. coli). Meanwhile, we also constructed and expressed the rM2e-ΔPly protein in E. coli. These proteins were administered intramuscularly to BALB/c mice. rM2e-ΔPly protein induces higher levels of humoral and cellular responses compared with their comprising protein mixture or rM2e alone. rM2e-ΔPly protein enhances the survival rate, reduces viral loads and inflammatory response in lung challenged with PR8. The serum induced by rM2e-ΔPly protein can protect against PR8 by passive immunity and cross-react with multiple M2e peptides derived from different IAV subtypes. After IAV and Streptococcus pneumoniae (S. pneumoniae) co-infection, rM2e-Ply protein can improve survival, lower viral and bacterial burdens, and diminish inflammatory response in the lungs. These results demonstrate that rM2e-ΔPly protein can significantly protect against influenza virus, IAV and S. pneumoniae co-infection, indicating that rM2e-ΔPly protein has the potential to become a universal influenza vaccine.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Flagellin/Virus-like Particle Hybrid Platform with High Immunogenicity, Safety, and Versatility for Vaccine Development.

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.

Matrix protein of Influenza A virus affects the membrane structure depending on protein concentration, amount of anionic lipids and pH gradient

The Influenza A virus is one of the most dangerous pathogens, which causes high mortality and epidemics. It relates to the Ortomyxoviridae family of enveloped RNA viruses, whose genetic material is coated by protein and lipid shells. Cell infection happens via endocytosis. The matrix protein M1 of the Influenza A virus is involved in all stages of the viral lifecycle. The protein scaffold disintegrates during the acidification of the medium in the endosome and assembles back at the inner side of the plasma membrane during the budding of daughter virions.

Bacteriophage T4 Vaccine Platform for Next-Generation Influenza Vaccine Development.

Developing influenza vaccines that protect against a broad range of viruses is a global health priority. Several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is sufficiently immunogenic to elicit complete protection, and vaccine platforms that can enhance immunogenicity and deliver multiple antigens are desperately needed. Here, we report proof-of-concept studies for the development of next-generation influenza vaccines using the bacteriophage T4 virus-like particle (VLP) platform. Using the extracellular domain of influenza matrix protein 2 (M2e) as a readout, we demonstrate that up to ~1,281 M2e molecules can be assembled on a 120 x 86 nanometer phage capsid to generate M2e-T4 VLPs. These M2e-decorated nanoparticles, without any adjuvant, are highly immunogenic, stimulate robust humoral as well as cellular immune responses, and conferred complete protection against lethal influenza virus challenge. Potentially, additional conserved antigens could be incorporated into the M2e-T4 VLPs and mass-produced in E. coli in a short amount of time to deal with an emerging influenza pandemic.

Computational Design and Analysis of a Multi-epitope Against Influenza A virus.

Influenza A viruses are among the most studied viruses, however no effective prevention against influenza infection has been developed. So, designing an effective vaccine against Influenza A virus is a critical issue in the field of medical biotechnology. For this reason, to combat this disease, we have designed a novel multi-epitope vaccine candidate based on the several conserved and potential linear B-cell and T-cell binding epitopes by using the in silico approach. This vaccine consists of an ER signal conserved sequence, the PADRE conserved epitope and two conserved epitopes of Influenza matrix protein 2. T-cell binding epitopes from Matrix protein 2 were predicted by in silico tools of epitope prediction. The selected epitopes were joined by flexible linkers and physicochemical properties, toxicity, and allergenecity were investigated. The designed vaccine was antigenic, immunogenic, and non-allergenic with suitable physicochemical properties and has higher solubility. The final multi-epitope construct was modeled, confirmed by different programs and the molecular interactions with immune receptors were considered. The molecular docking assay indicated the interactions with immune-stimulatory toll-like receptor 3 (TLR3) and major histocompatibility complex class I (MHCI). The HADDOCK and H DOCK servers were used to make docking analysis, respectively. The docking analysis indicated a strong and stable binding interaction between the vaccine construct with major histocompatibility complex (MHC) class I and toll-like receptor 3. Overall, the findings suggest that the current vaccine may be a promising vaccine to prevent Influenza infection.

Influenza Virus-like Particle (VLP) Vaccines Expressing the SARS-CoV-2 S Glycoprotein, S1, or S2 Domains.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic had brought disastrous consequences throughout the entire world. While several manufactured vaccines have been approved for emergency use, continuous efforts to generate novel vaccines are needed. In this study, we developed SARS-CoV-2 virus-like particles (VLPs) containing the full length of spike (S) glycoprotein (S full), S1, or S2 together with the influenza matrix protein 1 (M1) as a core protein. Successfully constructed VLPs expressing the S full, S1, and S2 via Sf9 cell transfections were confirmed and characterized by Western blot and transmission electron microscopy (TEM). VLP immunization in mice induced higher levels of spike protein-specific IgG and its subclasses compared to naïve control, with IgG2a being the most predominant subclass. S full and S1 immune sera elicited virus-neutralizing activities, but these were not strong enough to fully inhibit receptor–ligand binding of the SARS-CoV-2. Neutralizing activities were not observed from the S2 VLP immune sera. Overall, our findings revealed that S full or S1 containing VLPs can be developed into effective vaccines.

M2e-Based Influenza Vaccines with Nucleoprotein: A Review.

Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.

Conformational triggers associated with influenza matrix protein 1 polymerization

A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization. We studied the structural plasticity and oligomerization of M1NT in solution using NMR spectroscopy. We show that the isolated domain can be induced by sterol-containing compounds to undergo a conformational change and self-associate in a pH-dependent manner consistent with the stacked dimer oligomeric interface. Surface-exposed residues at one of the stacked dimer interfaces are most sensitive to sterols. Several perturbed residues are at the interface between the N-terminal subdomains and are also perturbed by changes in pH. The effects of sterols appear to be indirect and most likely mediated by reduction in water activity. The local changes are centered on strictly conserved residues and consistent with a priming of the N-terminal domain for polymerization. We hypothesize that M1NT is sensitive to changes in the aqueous environment and that this sensitivity is part of a mechanism for restricting polymerization to the membrane surface. Structural models combined with information from chemical shift perturbations indicate mechanisms by which conformational changes can be transmitted from one polymerization interface to the other.

Expression and purification of Brucella spp lumazine synthase decameric carrier in fusion to extracellular domain of influenza M2E protein

Brucella spp. Lumazine synthase enzyme, is a decameric protein carrier that display foreign antigens effectively in a polyvalent manner. The applied strategy using with this molecule results in a higher density of antigen and enhancing immunogenicity of peptide vaccines. In the current study, Brucella lumazine synthase (BLS) was applied for fusion with influenza matrix protein 2 ecto domain (M2E) as a foreign peptide. The primary studies were based on bioinformatics tools and the fusion was expressed and purified in following levels. Forming of decamer was confirmed by electrophoresis and western blotting techniques. Influenza matrix protein 2 was stably expressed at the 10 amino terminals of lumazine synthase. The purified fusion was injected to mice and immune responses were evaluated with indirect enzyme linked immunosorbent assay (ELISA) technique. According to ELISA results yield of purification process was 41% with ion exchange method and the protein was as a single band in Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE). The titer of immunized mice serum with decameric fusion of lumazine synthase and matrix protein (M2BL) was determined to be more than 1:32000 by indirect ELISA. The level of responses against matrix protein in decameric state of M2BL, was about 20% higher than monomer M2BL. Anti M2BL was cross reacted effectively with influenza M2E and in comparison with samples injected with adjuvant the level of antiM2E was similar. The results in this study confirms the role of multi copy presentation systems and applicability of BLS as an antigen carrier and adjuvant in designing peptide vaccines.

Rotavirus Inner Capsid VP6 Acts as an Adjuvant in Formulations with Particulate Antigens Only.

Novel adjuvants present a concern for adverse effects, generating a need for alternatives. Rotavirus inner capsid VP6 protein could be considered a potential candidate, due to its ability to self-assemble into highly immunogenic nanospheres and nanotubes. These nanostructures exhibit immunostimulatory properties, which resemble those of traditional adjuvants, promoting the uptake and immunogenicity of the co-administered antigens. We have previously elucidated an adjuvant effect of VP6 on co-delivered norovirus and coxsackievirus B1 virus-like particles, increasing humoral and cellular responses and sparing the dose of co-delivered antigens. This study explored an immunostimulatory effect of VP6 nanospheres on smaller antigens, P particles formed by protruding domain of a norovirus capsid protein and a short peptide, extracellular matrix protein (M2e) of influenza A virus. VP6 exhibited a notable improving impact on immune responses induced by P particles in immunized mice, including systemic and mucosal antibody and T cell responses. The adjuvant effect of VP6 nanospheres was comparable to the effect of alum, except for induction of superior mucosal and T cell responses when P particles were co-administered with VP6. However, unlike alum, VP6 did not influence M2e-specific immune responses, suggesting that the adjuvant effect of VP6 is dependent on the particulate nature of the co-administered antigen.

PH-dependent secondary structure propensity of the influenza A virus M2 cytoplasmic tail

The cytoplasmic C-terminal tail of the matrix protein 2 (M2) from influenza A virus has a well conserved sequence and is involved in interactions with several host proteins as well as the influenza matrix protein 1 (M1). Whereas the transmembrane domain of M2 has been well characterised structurally and functionally, high resolution information about the distal cytoplasmic tail is lacking. Here we report the chemical shifts of the cytoplasmic tail of M2 and the chemical shift perturbations at low pH and in the presence of membrane mimetics. The cytoplasmic tail residues are mostly disordered but an extended backbone conformation is adopted by the LC3 binding motif and the putative M1 interaction site has partial helical content with a small pH-dependence. The chemical shift assignments provide a basis for further investigations into interactions of the M2 cytoplasmic tail with viral and host cell factors.

Double-Layered M2e-NA Protein Nanoparticle Immunization Induces Broad Cross-Protection against Different Influenza Viruses in Mice.

The development of a universal influenza vaccine is an ideal strategy to eliminate public health threats from influenza epidemics and pandemics. This ultimate goal is restricted by the low immunogenicity of conserved influenza epitopes. Layered protein nanoparticles composed of well-designed conserved influenza structures have shown improved immunogenicity with new physical and biochemical features. Herein, structure-stabilized influenza matrix protein 2 ectodomain (M2e) and M2e-neuraminidase fusion (M2e-NA) recombinant proteins are generated and M2e protein nanoparticles and double-layered M2e-NA protein nanoparticles are produced by ethanol desolvation and chemical crosslinking. Immunizations with these protein nanoparticles induce immune protection against different viruses of homologous and heterosubtypic NA in mice. Double-layered M2e-NA protein nanoparticles induce higher levels of humoral and cellular responses compared with their comprising protein mixture or M2e nanoparticles. Strong cytotoxic T cell responses are induced in the layered M2e-NA protein nanoparticle groups. Antibody responses contribute to the heterosubtypic NA immune protection. The protective immunity is long lasting. These results demonstrate that double-layered protein nanoparticles containing structure-stabilized M2e and NA can be developed into a universal influenza vaccine or a synergistic component of such vaccines. Layered protein nanoparticles can be a general vaccine platform for different pathogens.

Intragastric delivery of recombinant Lactococcus lactis displaying ectodomain of influenza matrix protein 2 (M2e) and neuraminidase (NA) induced focused mucosal and systemic immune responses in chickens

Compounding with the problem of frequent antigenic shift and occasional drift of the segmented genome of Avian Influenza Virus (AIV), vaccines based on major surface glycoproteins such as haemagglutinin (HA) to counter heterosubtypic AIV infection in chickens remain unsuccessful. In contrast, neuraminidase (NA), the second most abundant surface glycoprotein present in viral capsid is less mutable and, in some instances, successful in eliciting inter-species cross-reactive antibody responses. However, without selective activation of B-cells and T-cells, the ability of NA to induce strong cell mediated immune responses is limited, thus NA based vaccines cannot singularly address the risk of virus escape from host defence. To this end, the highly conserved ectodomain of influenza matrix protein-2 (M2e) has emerged as an attractive cross-protective vaccine target. The present study describes the potential of recombinant Lactococcus lactis (rL. lactis) in expressing functional influenza NA or M2e proteins and conferring effective mucosal and systemic immune responses in the intestine as well as in the upper respiratory airways (trachea) of chickens. In addition, lavages collected from trachea and intestine of birds administered with rL. lactis expressing influenza NA or M2e protein were found to protect MDCK cells against avian influenza type A/PR/8/34 (H1N1) virus challenge. Although minor, the differences in the expression of pro-inflammatory cytokines gene transcripts targeted in this study among the birds administered with either empty or rL. lactis could be attributed to the activation of innate response by L. lactis.

A Universal Influenza Nanovaccine for "Mixing Vessel" Hosts Confers Potential Ability to Block Cross-Species Transmission.

Influenza A virus (IAV), a deadly zoonotic pathogen, poses a tremendous threat and burden to global health systems. Pigs act as "mixing vessel" hosts to support and generate new pandemic viruses. Preventing the spread of IAV in pigs effectively can delay or even block cross-species transmission. Universal vaccines based on the highly conserved ectodomain of influenza matrix protein 2 (M2e) have been widely reported, but have not been applied due to inadequate protection. Porcine circovirus type 2 (PCV2) causes immunosuppression and promotes swine influenza virus (SIV) infection. Here, M2e is inserted into capsid protein of PCV2 without burying the neutralizing epitopes and self-assembles to form a bivalent nanovaccine. Inoculation with the nanovaccine induces robust M2e- and PCV2-specific immune responses. The nanovaccine confers protection against lethal challenges of IAV from different species in mice, and significantly reduces SIV titers in pigs' respiratory tract and blocks SIV transmission. These results indicate that the nanovaccine is an economical and promising PCV2 and universal IAV bivalent vaccine, and it will synergistically and powerfully offer potential ability to block IAV cross-species reassortment and transmission.

How flu homes in on the nucleus

Influenza Influenza A virus (IAV) causes human epidemics annually and poses an ongoing threat for pandemics. The IAV genome is packaged in the viral capsid and, for successful infection, must be released into the cytosol and then imported into the nucleus for replication. Miyake et al. studied the process by which incoming viral ribonucleoproteins (vRNPs) that carry the viral genome are unpackaged during cell infection and find their way to the nucleus. They found that transportin 1 (TNPO1) associates with incoming capsids by binding to a TNPO1-specific nuclear localization signal in the influenza matrix protein (M1). TNPO1 promotes the removal of M1 from the surface of bundled vRNPs, which leads to bundle disassembly. The dissociated vRNPs then interact with the nuclear import machinery, which delivers them via the nuclear pore complex into the nucleus. Nat. Microbiol. 10.1038/s41564-018-0332-2 (2019)

Protective Immunity Induced by Incorporating Multiple Antigenic Proteins of Toxoplasma gondii Into Influenza Virus-Like Particles.

Virus-like particle (VLP) as a highly efficient vaccine platform has been used to present single or multiple antigenic proteins. In this study, we generated VLPs (multi-antigen VLPs, TG146) in insect cells co-infected with recombinant baculoviruses presenting IMC, ROP18, and MIC8 of Toxoplasma gondii together with influenza matrix protein 1 (M1) as a core protein. We also generated three VLPs expressing IMC, ROP18, or MIC8 together with M1 for combination VLPs (TG1/TG4/TG6). A total of four kinds of VLPs generated were characterized by TEM. Higher number of VLPs particles per μm2 were observed in multi-antigen VLPs compared to combination VLPs. Mice (BALB/c) were intranasually immunized with multi-antigen VLPs or combination VLPs and challenged with T. gondii tachyzoites (GT1) intraperitoneally. Compared to combination VLPs, multi-antigen VLPs showed significantly higher levels of CD4+ T cell, and germinal center B cell responses with reduced apoptosis responses, resulting in significant reduction on parasite burden. These results indicate that higher efficacy of VLPs generated by multi-antigen VLPs can induce significant reduction of parasite burden and better survival of mice than that by combination VLPs, providing important insights into vaccine design strategy for VLPs vaccine expressing multiple antigenic proteins.