United States of America, as Represented by the Secretary of Agriculture

James J Zhu

Marvin J Grubman

Teresa B De Los Santos

Fayna C Diaz-San Segundo

Marla J Koster

Manuel V Borca

Elizabeth A Bishop

Palaniappan Ramanathan

Disclosed herein is a recombinant adenovirus genome, said adenovirus genome comprising a heterologous nucleic acid inserted into a cloning site of said genome, said heterologous nucleic acid comprising: (a) a first nucleic acid sequence comprising an adenovirus tripartite sequence (e.g., SEQ ID NO:1) operably linked to a second nucleic acid sequence encoding an interferon (e.g., SEQ ID NO:2); (b) a third nucleic acid sequence comprising a bovine growth hormone polyA termination sequence operably linked to said second nucleic acid sequence (e.g., SEQ ID NO:3); (c) a fourth nucleic acid sequence comprising a porcine elongation factor 1-alpha (EF1&#x3b1;) promoter (e.g., SEQ ID NO:4); (d) a fifth nucleic acid sequence operably linked to said fourth nucleic acid sequence, said fifth nucleic acid sequence encoding a suppressor of cytokine signaling 1 (SOCS1) protein (e.g., SEQ ID NO:5). Furthermore, there is disclosed a method of producing interferon in an animal (e.g., swine).

Recombinant adenovirus-based interferon biotherapeutics in swine

A member of the bovine type III IFN family, boIFN-&#x3bb;3, was identified and characterized. We expressed boIFN-&#x3bb;3 using a recombinant replication defective human adenovirus type 5 (Ad5) and demonstrated antiviral activity against foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) in bovine cells in vitro. Cattle were inoculated with Ad5-boIFN-&#x3bb;3 followed by intradermolingual or aerosol FMDV challenge. Results demonstrated that the type III IFN family is conserved in bovines and that treatment of cattle with boIFN-&#x3bb;3 alone or in combination with IFN-&#x3b1; is able to confer delayed and reduced severity of FMD. Furthermore inoculation with Ad5-boIFN-&#x3bb;3 alone conferred full protection against aerosol challenge for at least 7 days after administration suggesting that type III IFN used in combination with FMD vaccines could fill one of the current gaps in emergency vaccination against FMDV.

Antiviral activity of bovine type III interferon against foot-and-mouth disease virus

Previously we have identified a conserved domain (SAP, for SAF-A/B, Acinus, and PIAS) in the foot-and-mouth disease virus (FMDV) leader (L) protein coding region that is required for proper sub-cellular localization and function. Mutation of isoleucine 55 and leucine 58 to alanine (I55A, L58A) within the SAP domain resulted in a viable virus that displayed a mild attenuated phenotype in cell culture, along with altered sub-cellular distribution of L and failure to induce degradation of the transcription factor nuclear factor kappa-B. Here we report that inoculation of swine and cattle with this mutant virus results in the absence of clinical disease, the induction of a significant FMDV-specific neutralizing antibody response, and protection against subsequent homologous virus challenge. Remarkably, swine vaccinated with SAP mutant virus are protected against wild type virus challenge as early as two days post-vaccination suggesting that a strong innate as well as adaptive immunity is elicited. This variant could serve as the basis for construction of a live-attenuated FMD vaccine candidate.

Recombinant live attenuated foot-and-mouth disease (FMD) vaccine containing mutations in the L protein coding region

NS4B is one of the non-structural proteins of classical swine fever virus. By using functional genetics, we have discovered, in the predicted amino acid sequence of NS4B of CSFV strain Brescia, a motif that resembles those found in the toll-like receptor (TLR) proteins, a group of host cell proteins involved in the development of anti-viral mechanisms. We have located the TLR motif in two groups of amino acid triplets at amino acid positions 2531-3 (residues IYK) and 2566-8 (residues VGI) of the CSFV NS4B glycoprotein. We have constructed a recombinant CSFV (derived from an infectious clone containing the genetic information of the highly virulent strain Brescia) containing amino acid substitutions in the three amino acid residues at positions 2566, 2567 and 2568, where the VGI triplet has been replaced by an AAA triplet inside the NS4B glycoprotein. The obtained virus, named NS4B-VGIv, was completely attenuated in swine, showing a limited ability in spreading during the infection in vivo. Although attenuated, NS4B-VGIv efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection.

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Niantic, CT, United States of America

James J Zhu