A heterologous expression cassette, DNA construct and vaccine composition for immunization against flavivirus and/or other pathogens. DNA constructs, recombinant viruses and vaccine compositions containing the recombinant viruses were obtained. This invention also concerns and provide an improved expression vector of the live-attenuated yellow fever 17D virus. Modifications in the expression cassette of heterologous proteins in the intergenic E/NS1 region of the yellow fever 17D vaccine virus, were made. The two new functional domains inserted in the expression cassette were (1) a coding sequence for the N-glycosylation motif, located between the NS1 N-terminal motif and the heterologous protein and (2) a sequence which promoted the proteolytic cleavage, or not, of the recombinant protein in such a way as to release it from its C-terminal containing the transmembrane domains and, consequently, from its association with the membrane of the endoplasmatic reticulum—ER.

Attenuated viruses and methods of designing them are disclosed. In one embodiment, there is disclosed an attenuated form of a virulent virus comprising an RNA encoding a viral protein or a nucleic acid sequence transcribable to said RNA, wherein the folding energy or structure of the RNA is changed at positions of evolutionarily conserved RNA structures with respect to that of said RNA encoding said viral protein in the virulent virus so as to bring about attenuation of the virus.

The invention generally relates to the development of immunogenic compositions comprising a DNA copy of at least one live attenuated plus-sense single-stranded RNA virus genome, such as the genome of a live-attenuated Zika vims (ZIKV), Japanese encephalitis virus (JEV) or yellow fever vims (YFV). The immunogenic compositions can be used for treating or conferring protective immunity against diseases related to plus-sense single-stranded RNA viruses, e.g. for affording prolonged immunoprotection against such viruses and protective immunity is conferred at low diseases, e.g., as low as 300 or 500 ng after administration of a single pLAV dosage.

Attenuated viruses and methods of designing them are disclosed. In one embodiment, there is disclosed an attenuated form of a virulent virus comprising an RNA encoding a viral protein or a nucleic acid sequence transcribable to said RNA, wherein the folding energy or structure of the RNA is changed at positions of evolutionarily conserved RNA structures with respect to that of said RNA encoding said viral protein in the virulent virus so as to bring about attenuation of the virus.

The present invention describes methods of generating a modified viral genome, producing infectious RNA, and generating modified viruses. The modified viral genome, infections RNA, and modified viruses comprise deoptimized nucleic acids; for example, codon-pair deoptimized or synonymous codon deoptimized. These modified viruses can be used in vaccines and methods of eliciting a protective immune response.

This disclosure provides ribonucleic acid (RNA) polynucleotides encoding replication-competent viral genomes that, when introduced to a subject, induce an active viral replication. The RNA may be provided naked or with an artificial RNA delivery system. The viral genome may be a full-length genome of an attenuated viral strain. For example, the RNA may encode an attenuated Chikungunya or yellow fever virus. The artificial RNA delivery system may be a lipid particle such as a lipid nanoparticle (LNP), a nanostructure lipid carrier (NLC), or a cationic nanoemulsion (CNE). This disclosure also provides methods of inducing an immune response, including protective immunity, by administering to a subject an RNA polynucleotide that encodes a replication-competent viral genome in an amount sufficient to cause viral replication in the subject. The immune response may include inducing the production of neutralizing antibodies at a level comparable to inoculation with a live-attenuated virus.

Described herein are methods for inactivation of viruses with high yield and recovery, and compositions produced by such methods.

This invention relates to a vaccine comprising live attenuated Zika virus comprising a partly codon deoptimized viral genome, a Zika virus comprising a partly codon deoptimized viral genome, as well as their use in methods of treatment and prevention of viral infection. is deoptimized along the nonstructural ZIKV coding region. In some embodiments, the non-structural region of the viral genome is codon deoptimized, and preferably one or more of the genes NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized.

The present invention provides an efficient process for culturing viruses in the presence of an endonuclease and for producing vaccines, typically from live attenuated viruses, under conditions to reduce the presence of host cell DNA and eliminate the need for a post-harvest DNA digestion step.

Compositions for protecting subjects from diseases caused by (+) SS RNA virus are described herein. The compositions include (i) a vector containing a DNA encoding a RNA molecule of an infectious (+) SS RNA virus operably linked to a eukaryotic RNA polymerase promoter and a carrier; or (ii) (+) SS RNA viruses obtained from eukaryotic cells transfected with the vector of (i) and a carrier.