The present disclosure provides isolated polynucleotides comprising multiple coding regions that are linked. In some aspects, a polynucleotide comprises at least a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first antigen and the second antigen are not the same, and wherein the first coding region and the second coding region are linked. The present disclosure also relates to the use of such polynucleotides to express multiple antigens in a cell and to induce multi-antigen-specific immune response in vivo.