Coxsackievirus vectors and their use in prevention and treatment of disease

#The present invention relates generally to the fields of molecular biology and virology. More specifically, the present invention relates to an attenuated Coxsackievirus, its use as a delivery vehicle for nucleic acids encoding antigenic or biologically active proteins, and treatment or prevention of viral infection or type 1 diabetes.

#Various scientific articles, scholarly publications and patent documents are referred to herein to describe the state of the art to which the invention pertains. Each of these documents is incorporated by reference herein in its entirety.

#The coxsackieviruses, members of the family Picornaviridae, are divided into two groups, based essentially on their pathogenicity and replication in newborn mice. The Group B coxsackieviruses (CVB) are composed of six serotypes (1-6). Coxsackievirus capsids are 29-nm-diameter icosahedral structures with the typical enterovirus canyon-like depressions surrounding the fivefold axes, which, by analogy to polioviruses and rhinoviruses, are binding sites for the cell membrane receptor human coxsackievirus adenovirus (Ad) receptor (HCAR).

#The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. The appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

#FIG. 1 shows the mIL4 insert in the CVB3/0-IL4 genome. The mIL4 sequence has been cloned between the viral capsid protein P1-D and the viral protease 2A (P2-A). During translation of the viral polyprotein, the most likely mechanism is that the protease P2-A cleaves itself out of the nascent protein in cis and cleaves the site between the capsid protein P1-D and mIL4 sequence in trans. Nucleic acid sequence on top line is SEQ ID NO:1; nucleic acid sequence on bottom line is SEQ ID NO:2; P2-A cleavage site is SEQ ID NO:9; mIL4 insert is SEQ ID NO:10.

#FIG. 2 shows the amino acid sequence of the PLS-CVB3 genome (SEQ ID NO:11) and the mIL-10-CVB3 (SEQ ID NO:12) at the site of the protease 2A cleavage. In this construct, the cloning procedure has been modified to include a polylinker site (PLS) to facilitate the use of the CVB3 as a generic cloning and expression vehicle. Further modifications include non-direct repeat genetic sequences to code for the protease P2-A cleavage site in the nascent polyprotein. The amino acids donated by the PLS are underlined, while the amino acids which form the 2A cleavage recognition signal are double underlined. The sequence of the mIL-10 insertion is shown in bold.

#The following definitions are used throughout the specification.

#As used herein, the term “Coxsackie B3 virus;” or “CVB33” refers to a specific serotype of the human coxsackie B enterovirus of the family Picornaviridae, genus Eterovirus. The CVB3 genome is characterized by a single molecule of positive sense RNA which encodes a 2,185 amino acid polyprotein.

#As used herein, the term “cardiotropic” refers to the targeting of heart tissue by a virus, in this case Coxsackievirus B3.