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.

Introduction

Influenza viruses cause both seasonal, epidemic infections and periodic, unpredictable pandemics. Vaccination is the most effective means to reduce the substantial morbidity and mortality caused by influenza infection [1], [2]. Hemagglutinin (HA), the major influenza surface antigen, binds host cell surface receptors and mediates viral entry by mediating membrane fusion [3], [4]. HA is the major target for virus-neutralizing antibodies and the most important antigen in subunit or split influenza vaccines (inactivated influenza vaccine-IIV) [5].

IIV potency is determined primarily by the quantification of immunologically active HA (able to elicit robust neutralizing or hemagglutination inhibiting [HI] antibody responses) that a dose contains. For IIV formulation, release, and stability testing, an in vitro potency test is used, single-radial immunodiffusion (SRID) [6], [7]. This modified Ouchterlony test quantifies HA based on the relative diameters of immunoprecipitin rings that form when vaccine antigen or homologous antigen standards diffuse radially from a circular well punched into an agarose gel that has been cast with a matched strain-specific sheep antiserum. The immunoprecipitin ring is detected by Coomassie blue staining after non-complexed antigen and antibody are removed by blotting with filter paper and washing with water. Although HA in IIVs form rosettes and other complexes, zwittergent is added to the antigen to disperse HA to smaller complexes such that the ring size is expected to be proportional to the HA concentration [7]. A weak correlation has been shown between SRID-measured vaccine potency and vaccine immunogenicity in clinical trials [8], [9], [10]. SRID has been accepted by international regulatory agencies and used by influenza vaccine manufacturers for IIV formulation, release, and stability testing for four decades.

Despite its selectivity for immunologically active HA, SRID has shortcomings. The most obvious shortcoming is the need for large quantities of strain-specific reference reagents, calibrated reference antigens and antisera. Generation and calibration of these strain-specific reagents is a time-consuming process that can delay vaccine release. The strain-specific HA reference antigens are produced by growing, inactivating, and purifying whole influenza viruses. The corresponding strain-specific antisera are generated by immunizing sheep multiple times with HA cleaved from purified whole virus by bromelain [6], [11]. For highly pathogenic pandemic strains, safety concerns can slow the production of both immunological reference reagents. Reference antigens and antisera are calibrated and distributed by the World Health Organization (WHO) Essential Regulatory Laboratories (ERLs). Primary liquid standards (PLS) are generated by assigning a value with physico-chemical methods to inactivated whole virus preparations. A conformationally independent total protein assay (BCA, Lowry, or Kjeldahl) is used to quantify the standard’s total protein content. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to determine the percent of HA contained in the sample through densitometry of the Coomassie blue stained bands [12], [13]. At this point, SRID is used to calibrate lyophilized standards which manufacturers will use against the relevant characterized PLS. PLS and reference standard are calibrated by the four ERLs in parallel. Thus, material shipment between geographically dispersed organizations, with diverse import regulations, can impose a significant barrier for timely pandemic responses [14]. The time required to generate and calibrate the reagents needed for SRID was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative in vitro potency assays that do not require strain-specific immunological reagents [13].

HA forms a membrane-bound trimer of disulfide linked HA1/HA2 heterodimers [15]. HA generally maintains a “metastable”, pre-fusion conformation at neutral pH, which elicits influenza neutralizing and HI antibodies. Once an energy barrier is overcome by an endosomal low pH drop, HA refolds extensively and irreversibly to a post-fusion conformation that is more stable, but significantly less immunologically active [16], [17], [18], [19]. HA conformation is also correlated with HA susceptibility to proteolysis [20], [21]. HA1 in pre-fusion HA, the more immunogenic form, is trypsin-resistant whereas HA1 in the less immunogenic, post-fusion HA form is trypsin-sensitive. This is consistent with the observation that native, well-folded protein domains are often protease resistant and stressed, denatured proteins are trypsin sensitive. Limited trypsin digestion (LTD) has been shown to be able to differentiate HA conformation [22], [23] and confer functional specificity to biophysical techniques that are generally insensitive to conformation without requiring strain-specific antibodies.

Biophysical techniques, such as reversed-phase high performance liquid chromatography (RP-HPLC) with either ultraviolet or fluorescence detection, have been developed and used for quantitative analysis of influenza HA [24], [25]. These methods measure the peak area of the HA1 and quantify it by comparing it to peak areas from reference standards. Like SRID, RP-HPLC still relies on the availability and reliability of the standardized reference antigens. RP-HPLC separates HA1 from other viral proteins based on their different hydrophobicity, but cannot distinguish HA1 between subtypes with similar hydrophobicity, therefore it cannot reliably quantitate subtype-specific HAs in multivalent vaccines.

Isotope dilution mass spectrometry (IDMS) is another biophysical technology recently developed for absolute quantification of HA in a vaccine mixture [26]. IDMS involves selecting specific target peptides from HA sequences as a stoichiometric representative of the intact protein. A known amount of synthetic reference peptide, in which one amino acid has been isotopically labeled, is spiked into the sample. Quantification is achieved by comparing the peak area of the isotopically labeled reference peptide with that of an endogenous target peptide generated by target protein proteolytic cleavage. Therefore, this approach can provide absolute HA quantification without the need for the time-consuming generation and calibration of strain-specific standardized reference antigens. In addition, since the method is based on sequence-specific peptides from each subtype, IDMS has been shown to be capable of selectively quantifying subtype-specific HAs in multivalent vaccines.

LTD functions as a pre-treatment to differentiate immunologically active HA without the need for sheep antisera. IDMS serves as an accurate biophysical approach to evaluate absolute HA quantification without the need of reference antigens. Here we report that we develop an LTD-IDMS method including LTD, IDMS and an intermediate biophysical step, precipitation, to separate LTD-resistant HA from LTD-digested HA to enable the specific, absolute quantification of immunologically active HA. We demonstrate that this biophysical alternative potency assay is able to provide potency measurements of pre-pandemic A/Shanghai/2/2013 (H7N9) vaccine that are comparable to SRID. Because the IDMS target peptides are conserved through the subtype, no method optimization was required since the peptides were first chosen for H7N2 and H7N7 [27]. Thus, the LTD-IDMS assay was rapidly applied for potency evaluation of the new H7N9 strain, A/Hong Kong/125/2017, before its strain-specific SRID reference reagents became available. The comparability study performed later confirmed that LTD-IDMS led to potency results comparable to SRID.