Reference antigen-free and antibody-free LTD-IDMS assay for influenza H7N9 vaccine in vitro potency determination

Abstract

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. Hemagglutinin (HA), as the main antigen in inactivated influenza vaccines (IIVs), elicits functional neutralizing antibodies and largely determines IIV effectiveness. HA potency has been evaluated by single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, to predict vaccine immunogenicity with a correlation to protective efficacy. We previously reported that limited trypsin digestion (LTD) selectively degraded stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique could specifically quantify trypsin-resistant, immunologically active HA. Here, we demonstrate that isotope dilution mass spectrometry (IDMS), a method capable of quantifying the absolute HA concentration without reference antigen use, can be further expanded by adding LTD followed with precipitation to selectively quantify the active HA. We test the LTD-IDMS assay on H7N9 vaccines stressed by low pH, raised temperature, or freeze/thaw cycles. This method, unlike SRID, has no requirement for strain-specific reference antigens or antibodies and can generate potency values that correlate with SRID. Thus, LTD-IDMS is a promising alternative in vitro potency assay for influenza vaccines to complement and potentially replace SRID in a pandemic when strain specific reagents may not be readily available.

Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry

“… 22 Mass spectrometry has also been employed extensively for viral protein characterization. Seasonal and pandemic influenza, with its substantial morbidity and mortality, has been a primary focus of many of these efforts, for antigen quantification, 23 − 28 vaccine potency determination, 29 , 30 and analysis of protein glycosylation. 31 − 34 As the COVID-19 pandemic has progressed, several reports on the use of mass spectrometry for the analysis of SARS-CoV-2 have been made.…”

Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material

“…This allowed rapid and accurate quantification of the absolute HA amount in the vaccine without the need for calibrated reference antigens. However, IDMS has a limited ability to distinguish between immunologically active versus inactive HA; LTD was developed to provide information about the conformation of the hemagglutinin protein. − To bridge LTD and IDMS and form the complete biophysical potency assay, precipitation was introduced post-LTD treatment to remove trypsin-digested HA1 peptides and retain trypsin-resistant native-conformation HA. As a result, precipitation enabled IDMS to quantify HA potency and mass in one single analytical runHA1 peptides quantified HA in its conformationally correct state, and HA2 peptides quantified total HA in the starting material.…”

“…LTD-IDMS was previously used to measure A­(H7N9) vaccine material that had been compromised by chemical or physical stressors . Here, we expand the method and demonstrate its ability to quantify the A­(H5N1) subtype without the need for strain-specific reagents and in the presence of MF59.…”

“…Each HA subcomponent has two sections that are external to the viral envelope: a globular head structure that is exclusively composed of HA1 residues and an elongated stem which is comprised of residues from both the HA1 and the HA2 regions. By quantifying representative peptide sequences from both the HA1 and the HA2 regions of the inactivated Influenza vaccine and then comparing that with an isotopically labeled peptide version of the same peptide, IDMS can provide an accurate total HA content. , IDMS as a stand-alone method does have one key limitation, however: it has a limited ability to distinguish between stressed and unstressed HA …”

“…HA that has experienced a decrease in pH or other stresses, however, undergoes unfolding in the HA1 region and is then susceptible to trypsin cleavage. Treating a vaccine sample that has experienced such a stress with limited tryptic digestion leads to digestion of the now exposed HA1 region, while the more protected HA2 region remains intact. − Protein precipitation separates the digested peptides from larger, intact, polypeptide pieces of the protein. Thus, the quantitative results obtained with LTD-IDMS using HA2 region peptides represent the total HA amount in the sample, while the results obtained using HA1 region peptides indicates the potent HA in the sample that generally correlates with the SRID results.…”