Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector

Abstract

The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

“…This includes fluorescent protein-encoding genes shown to be readily eliminated in cell culture or mice when present as NS1-fusion genes151620. Genetically stable reporter viruses offer several advantages, including the reliable identification and determination of the relative abundance of virus-infected cells by microscopic tracing of these cells in a model organism.…”

“…However, the integration of a reporter gene of this size represents a substantial increase of the overall segment length and is accompanied by substantial attenuation of viral replication11141516. As a consequence, especially viruses comprising the GFP gene were shown to lose reporter activity after passage in cell culture or mice151620, which could render them unfavorable for many experimental approaches, such as multicycle growth experiments, long-term infection of model organisms or transmission studies.…”

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice

“…The strategies for creating such vectors are diverse and comprise (i) expression of epitopes as parts of the hemagglutinin (HA) or (NA) [29], (ii) fusion to influenza virus genes [8,22], (iii) posttranslational cleavage, for example by 2A autocleavage sites [8,27] or caspase recognition sequences [18], (iv) reinitiation from a second reading frame by an overlapping stop-start codon [19] or IRES [12], (v) insertion of an additional reading frame by doubling the 3' non-codingregion (NCR) sequences [10,24] and (vi) expression from an artificial viral segment in addition to or instead of the viral genes [6,20,23,32,38,39]. Using influenza viruses as viral vectors has several advantages: First of all, their antigenic variability permits multiple prime-boost immunizations.…”

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

“… 8 Continuous passaging of the oncolytic IAV resulted in adaptive mutations throughout the viral genome, which led to enhanced stability and growth for both tumor and producer cell lines without affecting pathogenicity. 10 …”