mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

A bipartite NLS motif mediates the nuclear import of Drosophila moesin.

The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

Assessment of a quadrivalent nucleoside-modified mRNA vaccine that protects against group 2 influenza viruses.

Combined vaccine formulations targeting not only hemagglutinin but also other influenza virus antigens could form the basis for a universal influenza virus vaccine that has the potential to elicit long-lasting, broadly cross-reactive immune responses. Lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines can be utilized to efficiently target multiple antigens with a single vaccine. Here, we assessed the immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP vaccines that contain four influenza A group 2 virus antigens (hemagglutinin stalk, neuraminidase, matrix protein 2, and nucleoprotein) in mice. We found that all vaccine components induced antigen-specific cellular and humoral immune responses after administration of a single dose. While the monovalent formulations were not exclusively protective, the combined quadrivalent formulation protected mice from all challenge viruses, including a relevant H1N1 influenza virus group 1 strain, with minimal weight loss. Importantly, the combined vaccine protected from morbidity at a dose of 125 ng per antigen after a single vaccination in mice. With these findings, we confidently conclude that the nucleoside-modified mRNA-LNP platform can be used to elicit protection against a large panel of influenza viruses.

Parallel import mechanisms ensure the robust nuclear localization of actin in Drosophila.

Actin, as an ancient and fundamental protein, participates in various cytoplasmic as well as nuclear functions in eukaryotic cells. Based on its manifold tasks in the nucleus, it is a reasonable assumption that the nuclear presence of actin is essential for the cell, and consequently, its nuclear localization is ensured by a robust system. However, today only a single nuclear import and a single nuclear export pathway is known which maintain the dynamic balance between cytoplasmic and nuclear actin pools. In our work, we tested the robustness of the nuclear import of actin, and investigated whether the perturbations of nuclear localization affect the viability of the whole organism. For this aim, we generated a genetic system in Drosophila, in which we rescued the lethal phenotype of the null mutation of the Actin5C gene with transgenes that express different derivatives of actin, including a Nuclear Export Signal (NES)-tagged isoform which ensures forced nuclear export of the protein. We also disrupted the SUMOylation site of actin, suggested earlier to be responsible for nuclear retention, and eliminated the activity of the single nuclear import factor dedicated to actin. We found that, individually, none of the above mentioned manipulations led to a notable reduction in nuclear actin levels and thus, fully rescued lethality. However, the NES tagging of actin, together with the knock out of its importin, significantly reduced the amount of nuclear actin and induced lethality, confirming that the presence of actin in the nucleus is essential, and thereby, over-secured. Supporting this, we identified novel nuclear importins specific to actin, which sheds light on the mechanism behind the robustness of nuclear localization of actin, and supports the idea of essentiality of its nuclear functions.

Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses.

Messenger RNA (mRNA) vaccines represent a new, effective vaccine platform with high capacity for rapid development. Generation of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is a necessity for reducing influenza-associated morbidity and mortality. Here we focus on the development of a universal influenza B virus vaccine based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform. We evaluate vaccine candidates based on different target antigens that afford protection against challenge with ancestral and recent influenza B viruses from both antigenic lineages. A pentavalent vaccine combining all tested antigens protects mice from morbidity at a very low dose of 50 ng per antigen after a single vaccination. These findings support the further advancement of nucleoside-modified mRNA-LNPs expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine.

Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines have proven to be very successful in the fight against the coronavirus disease 2019 (COVID-19) pandemic. They are effective, safe, and can be produced in large quantities. However, the long-term storage of mRNA-LNP vaccines without freezing is still a challenge. Here, we demonstrate that nucleoside-modified mRNA-LNPs can be lyophilized, and the physicochemical properties of the lyophilized material do not significantly change for 12 weeks after storage at room temperature and for at least 24 weeks after storage at 4°C. Importantly, we show in comparative mouse studies that lyophilized firefly luciferase-encoding mRNA-LNPs maintain their high expression, and no decrease in the immunogenicity of a lyophilized influenza virus hemagglutinin-encoding mRNA-LNP vaccine was observed after 12 weeks of storage at room temperature or for at least 24 weeks after storage at 4°C. Our studies offer a potential solution to overcome the long-term storage-related limitations of nucleoside-modified mRNA-LNP vaccines.

The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila.

Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.

Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.

Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.

Nuclear actin: ancient clue to evolution in eukaryotes?

Until recently it was widely accepted that the dynamic cytoskeletal matrix is exclusive to the cytoplasm of eukaryotes, evolving before the emergence of the cell nucleus to enable phagocytosis, cell motility and the sophisticated functioning of the endomembrane system within the cytosol. The discovery of the existence of a prokaryotic cytoskeleton has changed this picture significantly. As a result, the idea has taken shape that the appearance of actin occurred in the very first cell; therefore, the emergence of microfilaments precedes that of the eukaryotic cytoskeleton. The discovery of nuclear actin opened new perspective on the field, suggesting that the nuclear activities of actin reflect the functions of primordial actin-like proteins. In this paper, we review the recent literature to explore the evolutionary origin of nuclear actin. We conclude that both ancient and eukaryotic features of the actin world can be detected in the nucleus today, which supports the idea that the cytoskeleton attained significant eukaryotic innovations before the tandem evolution of the cytoskeleton and nucleus occurred.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.