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Expression of Full-Length Neuraminidase (NA)
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Background:

Like hemagglutinin (HA), antibodies recognizing neuraminidase (NA) can provide both matched and cross-protection against influenza virus strains. In addition, NA also evolves and drifts independently of HA. These properties imply that improving the NA response might increase the breadth of the vaccine coverage and mitigate many of the yearly challenges that influenza vaccines face.

Problem/Rationale:

The expression of full-length neuraminidase has been a challenge due to its membrane-anchored nature, hydrophobicity, and a high number of cysteine and proline residues. Previous studies have shown expression of NA that was without transmembrane domain and was not membrane-anchored. Hence, it may have limited potential for a potential vaccine candidate vs. full-length NA.

Solution/Approach:

At Premas, we used D-Crypt™, the proprietary S. cerevisiae -based expression platform, to express membrane-anchored NA against influenza virus A.

The full-length gene was cloned into a proprietary episomal expression vector, pYRE100. The construct was then transformed into protease deficient proprietary S. cerevisiae host strain PYPD and expression scouting was performed using various parameters. This combination of the vector-host duo, an application of certain other vital parameters such as codon optimization, choice of Kozak sequence before ATG, and media composition using various carbon sources, have shown significantly high expression levels of NA. Expression and localization of NA were confirmed by immunoblotting, peptide mapping, and confocal microscopy (Figure 1), respectively. Sequencing confirmed the presence of 17 cysteines and 20 proline residues which was necessary for its correct conformation. We have further opportunities of doing codon harmonization in terms of strain engineering for the enhanced expression in S. cerevisiae and scale-ups.


Fig 1: Confocal microscopy demonstrating localization of NA at cell surface



Measurable Impact:

1) Expression of full-length NA 2) scale-up protein production up to 1100L for preclinical and clinical studies.
2) Premas is developing a VLP influenza flu vaccine candidate which will involve two other additional proteins .
3) Premas is looking to collaborate with potential partners for diagnostics, vaccine development, high-throughput drug screening and other usages of NA.



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