Quantification of viral proteins of the avian H7 subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic

Abstract

Vaccination is the most effective means to prevent influenza and its serious complications. Influenza viral strains undergo rapid mutations of the surface proteins hemagglutinin (HA) and neuraminidase (NA) requiring vaccines to be frequently updated to include current circulating strains. It is nearly impossible to predict which strains will be circulating in the next influenza season. It is, therefore, imperative that the process of producing a vaccine be streamlined and as swift as possible. We have developed an isotope dilution mass spectrometry (IDMS) method to quantify HA and NA in H7N7, H7N2, and H7N9 influenza. The IDMS method involves enzymatic digestion of viral proteins and the specific detection of evolutionarily conserved target peptides. The four target peptides that were initially chosen for analysis of the HA protein of H7N2 and H7N7 subtypes were conserved and available for analysis of the H7N9 subtype that circulated in China in the spring of 2013. Thus, rapid response to the potential pandemic was realized. Quantification of a protein is performed by employing multiple peptides to ensure that the enzymatic digestion of the protein is efficient in the region of the target peptides, verify the accuracy of the measurement, and provide flexibility in the case of amino acid changes among newly emerging strains. The IDMS method is an accurate, sensitive, and selective method to quantify the amount of HA and NA antigens in primary liquid standards, crude allantoic fluid, purified virus samples, and final vaccine presentations.

Figure 1

The amino acid sequences of the HA and NA proteins of the H7N7 strains and the NA of the H7N2 and the H7N9 proteins are shown with target peptides in bold and underlined. To ensure complete protein digestions, four unique H7 peptides were chosen from different regions of the HA protein. Obtaining similar results using at least two peptides located in different regions demonstrates that the protein is completely digested in the region of the peptides used for protein quantitation.

Figure 2

Amino acid sequence of strains of H7N7, H7N2, H7N3, and H7N9 showing the conserved peptides used for IDMS quantification of HA. The peptides chosen can be used to quantify strains collected from various areas of the world including China, Mexico, United States, and the Netherlands.

Figure 3

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) extracted ion chromatograms of target H7 and N7 peptides for quantifying a standard in which 6 peptide pairs were simultaneously monitored. Amino Acids shown in red are ¹³C and ¹⁵N stable isotope labeled. Data were acquired on a Thermo Scientific TSQ Vantage.