Better influenza vaccines: an industry perspective.

Vaccination is the most effective measure at preventing influenza virus infections. However, current seasonal influenza vaccines are only protective against closely matched circulating strains. Even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. Fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. This review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. Special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. The potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective.

Development of Pyrosequencing Assay for Evaluation of Genetic Stability of Vaccine Strains of Live Attenuated Influenza Type B Vaccine.

We developed a pyrosequencing protocol for monitoring of stability of attenuating mutations in the genome of vaccine reassortants based on master donor virus of Russian live attenuated influenza vaccine B/USSR/60/69. The developed protocol allows rapid and accurate assessment of mutations and can be used for analysis of genetic stability of reassortants during vaccine strain development and manufacturing, as well as genetic stability of vaccine isolates of influenza B virus during pre-clinical and clinical trials.

Quantification of Influenza Neuraminidase Activity by Ultra-High Performance Liquid Chromatography and Isotope Dilution Mass Spectrometry.

Mounting evidence suggests that neuraminidase's functionality extends beyond its classical role in influenza virus infection and that antineuraminidase antibodies offer protective immunity. Therefore, a renewed interest in the development of neuraminidase (NA)-specific methods to characterize the glycoprotein and evaluate potential advantages for NA standardization in influenza vaccines has emerged. NA displays sialidase activity by cleaving off the terminal N-acetylneuraminic acid on α-2,3 or α-2,6 sialic acid containing receptors of host cells. The type and distribution of these sialic acid containing receptors is considered to be an important factor in transmission efficiency of influenza viruses between and among host species. Changes in hemagglutinin (HA) binding and NA specificity in reassortant viruses may be related to the emergence of new and potentially dangerous strains of influenza. Current methods to investigate neuraminidase activity use small derivatized sugars that are poor models for natural glycoprotein receptors and do not provide information on the linkage specificity. Here, a novel approach for rapid and accurate quantification of influenza neuraminidase activity is achieved utilizing ultra-high performance liquid chromatography (UPLC) and isotope dilution mass spectrometry (IDMS). Direct LC-MS/MS quantification of NA-released sialic acid provides precise measurement of influenza neuraminidase activity over a range of substrates. The method provides exceptional sensitivity and specificity with a limit of detection of 0.38 µM for sialic acid and the capacity to obtain accurate measurements of specific enzyme activity preference toward α-2,3-sialyllactose linkages, α-2,6-sialyllactose linkages, or whole glycosylated proteins such as fetuin.

Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis µ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

Analysis of the proficiency of single radial immunodiffusion assays for quality control of influenza vaccines in Korea.

Influenza vaccine potency, which is determined by quantitatively measuring the content of Hemagglutinin (HA), is an essential index representing the efficacy of the vaccine. Standardization of the single radial immunodiffusion (SRID) assay, a method for measuring HA content, and proficiency of the testing institutions are crucial for influenza vaccine quality control. Herein, we assessed the proficiency of SRID assays at the National Control Laboratory (NCL) of Korea and several vaccine manufacturers. Eight laboratories participated in this study, and the proficiencies of all laboratories yielded satisfactory results in overall SRID assays. In contrast, there were some unsatisfactory results in measuring with different types of agarose gel plates produced by other laboratories. Overall, our findings demonstrated that the proficiency of SRID assay in the tested laboratories is acceptable for quality control of influenza vaccines and that detailed review on the validation reports regarding the test methods will be helpful for better control.

[Infectivity Titers of Each Component of the Influenza Virus in the Live Vaccine Purchased from a Parallel Import Distributing System].

Currently in Japan, the only approved influenza vaccine is the inactivated vaccine which is injected subcutaneously. On the other hand, there is a live vaccine available elsewhere in the world. Flumist, an intranasal influenza live vaccine which contains four strains of infectious viruses, has been used in the United States for more than 10 years; the vaccine has been found effective in clinical trials, while it has some limitations such as those on subjects for the administration, strict storage conditions, relatively short expiration date etc. It is not yet approved in Japan, but available through personal import by some medical institutions, and prescribed based on the decision of the doctor. However, in Japan, there is no checking system whether the vaccine contains appropriate amounts of infectious viruses or not. In the present study, we purchased 2013-14 and 2014-15 years' lots of Flumist from a parallel importer and measured the amount of infectious viruses of each component of them using the focus assay. Consequently, for type A influenza viruses, the titers of both of H1N1pdm09 and H3N2 viruses in the 2013-14's lot were 1/30 of the lower limit of those shown in the package insert and 1/10 in 2014-15's lot, while those of type B viruses, both of B/Massachusetts and B/Brisbane viruses marginally cleared the lower limit. The digital PCR analysis showed that the absolute genome copy numbers of type A viruses were 1/10 of those of type B viruses. The relatively higher titer of B/Massachusetts also gradually decreased over time during its storage at 4°C and finally reached the lower limit at about one week before the expiration date. In case it is approved officially in the future to be used in Japan, some studies will be required to elucidate the minimum viral titers of the components necessary for effective live vaccine. In addition, there should be a system to check the titer during the distribution process in Japan.

Impact of PM2.5 and ozone on incidence of influenza in Shijiazhuang, China: a time-series study.

Most of the studies are focused on influenza and meteorological factors for influenza. There are still few studies focused on the relationship between pollution factors and influenza, and the results are not consistent. This study conducted distributed lag nonlinear model and attributable risk on the relationship between influenza and pollution factors, aiming to quantify the association and provide a basis for the prevention of influenza and the formulation of relevant policies. Environmental data in Shijiazhuang from 2014 to 2019, as well as the data on hospital-confirmed influenza, were collected. When the concentration of PM2.5 was the highest (621 µg/m3), the relative risk was the highest (RR: 2.39, 95% CI: 1.10-5.17). For extremely high concentration PM2.5 (348 µg/m3), analysis of cumulative lag effect showed statistical significance from cumulative lag0-1 to lag0-6 day, and the minimum cumulative lag effect appeared in lag0-2 (RR: 0.760, 95% CI: 0.655-0.882). In terms of ozone, the RR value was 2.28(1.19,4.38), when O3 concentration was 310 µg/m3, and the RR was 1.65(1.26,2.15), when O3 concentration was 0 µg/m3. The RR of this lag effect increased with the increase of lag days, and reached the maximum at lag0-7 days, RR and 95% CI of slightly low concentration and extremely high concentration were 1.217(1.108,1.337) and 1.440(1.012,2.047), respectively. Stratified analysis showed that there was little difference in gender, but in different age groups, the cumulative lag effect of these two pollutants on influenza was significantly different. Our study found a non-linear relationship between two pollutants and influenza; slightly low concentrations were more associated with contaminant-related influenza. Health workers should encourage patients to get the influenza vaccine and wear masks when going out during flu seasons.

Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine.

Live attenuated influenza vaccines (LAIVs) targeting seasonal influenza are produced in embryonated eggs and formulated as a trivalent preparation of three live attenuated vaccine strains, one A(H1N1) strain, one A(H3N2) strain and one type B strain. In this study, we describe an egg-based potency assay for estimating the 50% Egg infectious dose (EID50) of individual strains in the trivalent preparation by selective neutralisation of two strains and then estimating the infective titres of the non-neutralised strain. The test is highly specific, and no cross interference of heterologous antisera is observed in the estimation of individual titres. Individual strains with titres in the range of 6.5-7.0 log EID50 per 0.5 ml show intra-assay and inter-assay coefficients of variance ranging from 1.25% to 2.95%. This assay was developed to establish a simple, reliable and inexpensive egg-based assay for estimating the potency of individual strains in a trivalent preparation.

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin.

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.

An Egg-Derived Sulfated N-Acetyllactosamine Glycan Is an Antigenic Decoy of Influenza Virus Vaccines.

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess different glycosylation patterns than viruses circulating among humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived glycan that is an antigenic decoy, with egg-binding MAbs reacting with a sulfated N-acetyllactosamine (LacNAc). Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken-derived cells, suggesting that only egg-grown vaccines can induce antiegg antibodies. Notably, antibodies against the egg antigen utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and had a simple heavy-chain complementarity-determining region 3. By analyzing a public data set of influenza virus vaccine-induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.