Described are methods for large-scale production of recombinant adenovirus 26, utilizing perfusion systems and infection at very high cell densities.

The present invention relates to the field of CAdV vector vaccines, and especially to promoters suitable to express target antigens from such vector vaccines. Disclosed and claimed are recombinant canine adenoviruses, methods of making them, uses for them (including in immunological, immunogenic, vaccine or therapeutic compositions, or, as a vector for cloning, replicating or expressing DNA and methods of using the compositions and vector), expression products from them, and uses for the expression products. Additionally, disclosed and claimed are truncated EHV4 promoters, expression cassettes containing the promoters, and recombinant viruses and plasmids containing the promoters or expression cassettes.

A method of using a cell to produce virus is provided involving growing cells under hyperosmotic conditions during a growth phase of the cells, infecting or transfecting the cells grown under hyperosmotic conditions with a virus, and maintaining the infected or transfected cells under less stressful conditions during a production phase of the infected or transfected cells to produce more of the virus. Viral productivity is improved by this method.

Methods of purifying adenovirus that can be performed on a large scale. The methods purify adenovirus from an adenovirus-containing sample comprising or derived from a host cell population by clarifying the sample, wherein clarification comprises depth filtration followed by microfiltration; processing the clarified sample by anion exchange chromatography; and processing the anion exchange product by tangential flow filtration (TFF) to provide a TFF product.

The present invention relates to a method for virus purification. The present invention provides downstream processes for purification of adenovirus from cell culture harvest. More closely, it relates to a method for adenovirus purification using a virus capture and a virus polishing step.

An adenovirus expression vector is provided. The adenovirus expression vector may include: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA is provided. A method of synthesizing an adenovirus vector is also provided. The synthesis may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub-fragment comprising a portion of the full genome of the adenovirus; b) circularizing the sub-fragments to form plasmid structures; and c) assembling the circularized sub-fragments into a linear structure, wherein the vector comprises a combination of two or more sub-fragments. A mammalian cell line configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products but is devoid of other adenovirus sequences is also provided.

Methods of purifying an adenovirus from an impure preparation are provided. In some embodiments, a combination of mixed mode chromatography and anion exchange chromatography is used to purify the adenovirus.

Embodiments herein relate to compositions of and methods for live viruses. In certain embodiments, a live, attenuated virus composition includes, but is not limited to, one or more live, attenuated viruses and compositions to reduce inactivation and/or degradation of the live, attenuated virus. In other embodiments, the live, attenuated virus composition may be a vaccine composition. In yet other compositions, a live, attenuated virus composition may include at least one carbohydrate, at least one protein and at least one high molecular weight surfactants for reducing inactivation and/or degradation of the live, attenuated virus.

Methods of purifying an adenovirus from an impure preparation are provided. In some embodiments, a combination of mixed mode chromatography and anion exchange chromatography is used to purify the adenovirus.

Recombinant adenovirus genomes that include an exogenous open reading frame (ORF) and a self-cleaving peptide coding sequence are described. Optimal placement of the exogenous genes for minimal impact on viral kinetics is further disclosed. Therapeutic applications of the recombinant adenoviruses are also described.