Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus

Abstract

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

“…2C). Our results, together with those of previous studies (10), reiterated that genome plasticity is a common characteristic among flaviviruses, especially for E (15)(16)(17) and NS1 (18), in which heterologous insertions into their intragenic regions are evidently tolerated to create fusion proteins that require no assistance from the inclusion of engineered flanking sequences recognized and cleaved by viral or cellular proteases. These findings strongly suggested that the C-terminal end of JEV NS1 is likely to be an inherently plastic domain in flaviviruses that has evolved to cope with a rapidly changing host environment.…”

“…Moreover, to stabilize such an insertion, this recombinant YFV appeared to further acquire additional compensatory mutations located in the E, NS4B, and NS1 proteins (18). Using a trans-complementation assay, we discovered in this study that the C-terminal end of JEV NS1 was sufficiently flexible to house heterologous epitopes without a significant impact on virus viability.…”

“…Using a direct transposon-mediated random-insertion technique, YFV NS1 was previously found to have only one position, at residue 236, that could accommodate a 56-aa epitope derived from influenza virus M2e, and the resulting recombinant virus, although viable, was completely impaired in terms of its dimerization capability (18). Moreover, to stabilize such an insertion, this recombinant YFV appeared to further acquire additional compensatory mutations located in the E, NS4B, and NS1 proteins (18).…”

“…Based on an assessment by three-dimensional (3D) structure modeling, the fg loop of the YF-17D E protein was previously found to be able to successfully accommodate a 9-amino-acid (aa) T-cell epitope (15) or a 16-aa B-cell epitope (16,17) from Plasmodium yoelii. By transposon-mediated insertion mutagenesis (18), a single insertion site at amino acid residue 236 of YF-17D NS1 was identified, where a 56-aa M2e epitope from influenza A virus can be inserted into NS1 to form a fusion protein. Nevertheless, such NS1 fusion proteins inevitably lose their dimerization capability compared with wild-type NS1 during virus infection.…”

The yellow fever 17D virus as a platform for new live attenuated vaccines

“…8,22,59 Another possibility for epitope expression was developed by means of a transposon-mediated technique to randomly insert and express influenza A virus epitopes into the yellow fever virus NS1 protein. 63 Intriguingly, by selecting recombinant viruses with a methodology combining plaque purification with immunostaining, only one single insertion site was obtained at NS1-236 in which the influenza A epitope was expressed, strongly suggesting that NS1 is not tolerant in terms of alternative insertion points. Immunization of mice with this recombinant virus was not sufficient to afford protection against challenge with the mouse-adapted influenza A/H1N1 virus.…”