Provided herein is an isolated polynucleotide, which encodes alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and a polypeptide comprising a coronavirus protein fused to a signal sequence and/or transmembrane domain. The coronavirus protein may be the receptor binding domain of the S1 subunit of coronavirus spike (S) protein. The polynucleotide such as RNA is useful for as a vaccine against coronavirus infection, especially, COVID-19 infection.

A novel and improved vaccine for prevention of disease caused by the Severe Acute Respiratory Syndrome-2 (SARS-2), /COVID-19 virus. Current mRNA and Adenovirus vaccine technologies for SARS-2 provide high levels of serum Immunoglobin G (IgG), antibodies against the original Wuhan strain, but there are now hundreds of mutant strains which can evade both vaccine and convalescent antibodies. These vaccines also do not provide strong mucosal IgA class antibodies which provide wider protection against mutant strains of Flu A and other respiratory viruses. The ability of these technologies to provide high levels of protection is in question, as serum neutralizing antibodies may decline to undetectable levels after six months. The appearance of mutant strains such as the Beta, Gamma, Delta, and Epsilon strains, containing altered amino acid sequences capable of evading vaccine-induced antibodies, calls for new vaccine technologies that can be quickly altered to meet this threat. The following describes a combination approach to prevention of infection by SARS-2/COVID-19. This combination consists of a priming injection of Recombinant Replicative Particles (VRP) derived from the Alphavirus Venezuelan Equine Encephalitis (VEE) strain 3000/3526, with insertion of a Delta/B.1.617.2 SARS-2/COVID-19 spike 1 glycoprotein (gp)-Receptor-Binding Domain (RBD) gene. The insertion of Internal Ribosome Entry Sites (IRES), elements between the 26S promoter and the SARS-2/COVID RBD gene allows for more efficient translation of the SARS-2/COVID gene products. The VEE.sup.3000/3256 VRP are produced from plasmids, so while they are infectious for one replicative cycle in vivo, progeny VRP are replication incompetent. The priming is followed by one or more intranasal administrations of a suspension of recombinant SARS-2/COVID-19 envelope spike 1 glycoproteins (gp), from selected mutant strains, combined with the pulmonary surfactant adjuvant, SF-10. The goal of the invention is to safely provide multiple immune layers of protection in both the upper and lower respiratory tracts, with induction of both mucosal IgA and serum IgG antibodies, as well as effector Cytotoxic T Lymphocyte (CTL), cells recognizing conserved regions of the SARS-2/COVID-19 virus genome. Secondary goals are to reduce the risk of antibody-dependent enhancement (ADE), of infection, a major concern with other SARS-2/COVID-19 vaccine designs, and to provide capacity to protect against mutant emergent strains of SARS-2/COVID-19 with annual intranasal boosters of new spike glycoproteins.

The present invention provides modified alphaviruses and compositions, methods, and kits for preparing and using them to elicit an immune response to an alphavirus in a subject.

The present invention generally relates to systems and methods suitable for high-level protein production. While one or more elements of the present invention are derived from an alphavirus, the present invention does not require propagation of virus particles. In particular, a system comprising two separate RNA molecules is foreseen, each comprising a nucleotide sequence derived from an alphavirus: one RNA molecule comprises a RNA construct for expressing alphavirus replicase, and one RNA molecule comprises a RNA re pi icon that can be replicated by the replicase in trans. The RNA construct for expressing alphavirus replicase comprises a 5-cap. It was surprisingly found that the 5-cap is suitable for efficiently driving expression of a transgene from the replicon in trans. The system of the present invention enables expression of a protein of interest in a cell or organism, but is not associated with undesired virus-particle formation. Therefore, the present invention is suitable for efficiently and safely producing a protein of interest, e.g. a therapeutic protein or an antigenic protein, such as a vaccine, in a target organism. Respective methods of protein production in vitro and in vivo as well as medical uses are provided herein. The present invention also provides DNA encoding the RNA molecules of the invention, and cells comprising the RNA molecules of the invention.

The present invention provides modified alphaviruses and compositions, methods, and kits for preparing and using them to elicit an immune response to an alphavirus in a subject.

The present invention relates to polynucleotides and alphaviral vectors for the expression of genes of interest in mammalian cells. Additionally, the invention relates to cells which comprise said polynucleotides and alphaviral vectors and are capable of stably expressing one or more genes of interest. The invention also relates to methods for obtaining said cells, to methods for expressing a gene of interest in said cells, and to methods for replacing the gene of interest stably expressed by said cells with another gene of interest.

Disclosed in the present invention are an RNA replicon for improving gene expression, and a use thereof. The RNA replicon comprises: 5 and 3 untranslated regions; a non-structural protein gene coding region, a subgenomic promoter and a target gene coding region. In the present invention, a PCR site-directed mutagenesis technique is used to introduce the non-structural protein region mutant replicable RNA, which is transfected into mammalian eukaryotic cells by means of Lipofectamine 2000 or nanoparticles, so as to significantly enhance the expression of cytokines and chemokines including GM-CSF, IFN-, IL-2, IL-12, IL-15 mediated by downstream subgenomic promoters, and can be applied to treatment of tumors, infectious diseases, autoimmune diseases, hereditary diseases or cardiovascular diseases.

The present disclosure relates to the field of molecular virology, including nucleic acid molecules comprising modified viral genomes or replicons (e.g., self-replicating RNAs), pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for eliciting an immune response in a subject in need thereof, as well as methods for preventing and/or treating various health conditions.

Provided herein is an isolated polynucleotide, which encodes alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and a polypeptide comprising a coronavirus protein fused to a signal sequence and/or transmembrane domain. The coronavirus protein may be the receptor binding domain of the S1 subunit of coronavirus spike (S) protein. The polynucleotide such as RNA is useful for as a vaccine against coronavirus infection, especially, COVID-19 infection.

The present invention relates to systems and methods suitable for high-level protein production. In particular, a system comprising two separate RNA molecules is foreseen, each comprising a nucleotide sequence derived from an alphavirus: one RNA molecule comprises a RNA construct for expressing alphavirus replicase, and one RNA molecule comprises a RNA replicon that can be replicated by the replicase in trans. The system of the present invention enables expression of a protein of interest in a cell or organism, but is not associated with undesired virus-particle formation. The present invention is suitable for efficiently and safely producing a protein of interest in a target organism. Respective methods of protein production in vitro and in vivo as well as medical uses are provided herein. The present invention also provides DNA encoding the RNA molecules of the invention, and cells comprising the RNA molecules of the invention.