Downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza A virus vaccines

Abstract

Vaccination represents the best option to protect humans against influenza virus. However, improving the effectiveness of current vaccines could better stifle the health burden caused by viral infection. Protein synthesis from individual genes can be downregulated by synthetically deoptimizing a gene's codon usage. With more rapid and affordable nucleotide synthesis, generating viruses that contain genes with deoptimized codons is now feasible. Attenuated, vaccine-candidate viruses can thus be engineered with hitherto uncharacterized properties. With eight gene segments, influenza A viruses with variably recoded genomes can produce a spectrum of attenuation that is contingent on the gene segment targeted and the number of codon changes. This review summarizes different targets and approaches to deoptimize influenza A virus codons for novel vaccine generation.

Keywords: codon deoptimization; codon usage bias; codon-pair bias; inactivated influenza vaccines; influenza A virus; influenza reverse genetics; live attenuated influenza virus; recombinant influenza vaccine; synthetic attenuated virus engineering.

Figure 1. Influenza A virus genome organization and flexibility

The eight influenza A virus segments are represented in 3′ to 5′ negative sense. Regions of flexibility are determined (white boxes) by areas inside ORFs that do not have cis-acting RNA elements (packaging signals and splice sites) or alternative overlapping ORFs. Noncoding regions are indicated in black boxes. Packaging signals are indicated in gray boxes. In frame (pink) and out of frame (red) overlapping ORFs are also indicated. Red circle indicates ribosomal slippage sequence. ORF: Open reading frame.

Figure 2. Codon bias of Homo sapiens and influenza A virus

A codon chart with the relative synonymous codon usage is displayed. Relative synonymous codon usage values (data taken from [50]) and frequency per 1000 amino acids was represented as percent per amino acid. For consensus, 83,487 Homo sapiens and 40,413 influenza A virus coding sequences were evaluated.

Figure 3. Proposed mechanism of attenuation for codon deoptimized influenza A viruses

(A) Schematic representation of wild-type influenza infection in a permissive cell. First, vRNP-mediated replication and transcription occurs in the cell nucleus, followed by viral protein synthesis (shown here as the HA and NA glycoproteins, and the IFN antagonist NS1), export of vRNPs from the nucleus (indicated by arrow) and production of infectious progeny (black virions). (B) During PR8^3F infection (light gray virions), less viral genome replication and gene transcription mediated by PB1 and NP deoptimization leads to a decrease in all viral proteins and their functions, including influenza NS1, nuclear export of vRNPs and virion production. (C) A paucity of influenza glycoproteins during (NA + HA)^Min infection (dark gray virions) results in ample viral protein production but minimal virion formation and release because of decreased levels of IAV HA and NA. (D) Infection with PR8 NScd (white virions) stimulates type I IFN responses because of suboptimal NS1 levels, and inefficient nuclear export of vRNPs due to low NEP levels. For simplicity, only certain stages of the life cycle are represented. IAV: Influenza A viruses; vRNP: Viral ribonucleoprotein.