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.

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.

Quantitation of residual valproic acid in flu vaccine drug substance.

Quantitative measurement of process-related impurities is a critical safety requirement for the production of drug substances of vaccine and therapeutic biologics. A simple and sensitive HPLC method has been developed for separation and quantitation of residual valproic acid (VPA) used in the cell transfection procedure for the manufacturing of an influenza vaccine. The method is comprised of a modified Dole liquid phase extraction followed by a quick pre-column derivatization using 2-bromoacetophenone. Nonanoic acid (NNA) is used as the internal standard (IS) and the quantification is performed by reversed-phase liquid chromatography. This new method can accurately measure as low as 6.8 µg/mL (LOQ) residual VPA in the vaccine drug substance.

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.

Application of stable isotope dimethyl labeling for MRM based absolute antigen quantification of influenza vaccine.

Determining the precursor/product ion pair and optimal collision energy are the critical steps for developing a multiple reaction monitoring (MRM) assay using triple quadruple mass spectrometer for protein quantitation. In this study, a platform consisting of stable isotope dimethyl labeling coupled with triple-quadruple mass spectrometer was used to quantify the protein components of the influenza vaccines. Dimethyl labeling of both the peptide N-termini and the ϵ-amino group of lysine residues was achieved by reductive amination using formaldehyde and sodium cyanoborohydrate. Dimethylated peptides are known to exhibit dominant a1 ions under gas phase fragmentation in a mass spectrometer. These a1 ions can be predicted from the peptide N-terminal amino acids, and their signals do not vary significantly across a wide range of collision energies, which facilitates the determination of MRM transition settings for multiple protein targets. The intrinsic a1 ions provide sensitivity for acquiring MRM peaks that is superior to that of the typical b/y ions used for native peptides, and they also provided good linearity (R2 ≥ 0.99) at the detected concentration range for each peptide. These features allow for the simultaneous quantification of hemagglutinin and neuraminidase in vaccines derived from either embryo eggs or cell cultivation. Moreover, the low abundant ovalbumin residue originated from the manufacturing process can also be determined. The results demonstrate that the stable isotope dimethyl labeling coupled with MRM Mass spectrometry screening of a1 ions (i.e., SIDa-MS) can be used as a high-throughput platform for multiple protein quantification of vaccine products.

Vacunación frente a la gripe estacional en las personas mayores. Evaluación de la vacuna tetravalente. Informe de posicionamie / Seasonal flu vaccination for older people: Evaluation of the quadrivalent vaccine. Positioning repor

La gripe es un importante problema de salud pública, particularmente en las personas susceptibles de presentar complicaciones asociadas, personas mayores, niños menores de 2 años, enfermos crónicos, inmunocomprometidos y embarazadas. Pero, además, la gripe tiene un gran impacto sanitario con un aumento de la demanda asistencial y un espectacular aumento de las visitas ambulatorias, sobrecargando los servicios de urgencias y hospitalarios. Durante los brotes epidémicos, las tasas de hospitalización de las personas mayores de 65 años son máximas y la mortalidad notificada por gripe en la temporada 2017/2018 ha sido de 960 defunciones. La vacunación antigripal estacional es el método con una mayor relación coste-efectividad de prevención primaria de la gripe, reduciendo las enfermedades respiratorias relacionadas, el número de visitas a las consultas médicas, el número de hospitalizaciones y muertes en personas de alto riesgo y el absentismo laboral en adultos. En los últimos años la gripe B ha recibido escasa atención en la literatura científica y, sin embargo, en períodos interepidémicos, la gripe B puede ser una de las principales causas de epidemias de gripe estacional, causando una considerable morbimortalidad y un aumento de costes. La vacuna tetravalente, a diferencia de la trivalente, obtiene una protección inmunológica frente al segundo linaje de la gripe B y, de acuerdo con una revisión crítica de la literatura científica, proporciona una protección más amplia sin afectar a la inmunogenicidad de las otras 3 cepas vacunales comunes a las vacunas trivalente y tetravalente. La vacuna tetravalente es coste-efectiva al disminuir el número de casos de gripe y siempre es una intervención rentable, con un importante ahorro de coste para el sistema de salud y para la sociedad, disminuyendo las tasas de hospitalización y de mortalidad asociadas a las complicaciones de la gripe

Influenza is a significant health problem, particularly in those persons susceptible to having associated complications, older people, children less than 2 years, patients with chronic diseases, immunocompromised patients, and pregnant women. But influenza also has a large impact on the health system, with an increase in the healthcare demand and a spectacular increase in outpatient visits, overloading the emergency and hospital services. During epidemic outbreaks, the hospital admission rates of people over 65 years are at a maximum, and the mortality notified for the 2017/2018 influenza season was 960 deaths. The seasonal anti-influenza vaccine is the method with a better cost-effective ratio of primary prevention of influenza, reducing associated respiratory diseases, the number of hospital admissions, and deaths in high risk individuals, as well as work absenteeism in adults. In the last few years, influenza B has received little attention in the scientific literature, although in the periods between epidemics influenza B can be one of the main causes of seasonal epidemics, causing considerable morbidity and mortality and an increase in costs. The quadrivalent vaccine has a second-line immunological protection against influenza B, and according to a critical review of the scientific literature, it provides wider protection without affecting immunogenicity of the other three vaccine strains common to the trivalent and tetravalent vaccine. The quadrivalent vaccine is cost-effective in reducing the number of influenza cases, and is always a worthwhile intervention, with a significant cost saving for the health system and for society, by reducing the hospital admission rates and mortality associated with the complications of influenza

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.

An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations.

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.

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.

Vacunación frente a la gripe estacional en las personas mayores. Evaluación de la vacuna adyuvada. Informe de posicionamiento / Seasonal flu vaccination for older people: evaluation of the adjuvanted vaccine. Positioning report

La gripe es un importante problema de salud pública, particularmente en las personas mayores, con una significativa carga clínica y económica y con una alta mortalidad. En España, durante la temporada 2015- 2016, se han notificado 3.101 casos graves hospitalizados confirmados por gripe, de los que han fallecido el 11% (352 casos). Además, hay un gran aumento de costes económicos y sanitarios por sus complicaciones y los mayores de 65 años representan aproximadamente el 64% del total de costes de la gripe. La vacuna antigripal estacional es la estrategia fundamental, y los estudios de coste-beneficio y coste-efectividad así lo demuestran. Uno de los objetivos prioritarios es mejorar la respuesta inmune de las vacunas, y una línea importante de investigación es la búsqueda e inclusión en las vacunas de adyuvantes o inmunoestimuladores. En este informe de posicionamiento se evalúa la vacunación en las personas mayores y la importancia de la vacuna adyuvada en los mayores, que refuerza la inmunogenicidad mediante una revisión crítica de la bibliografía relacionada con la mayor evidencia disponible sobre su inmunogenicidad, efectividad y evaluación económica (AU)

Flu is a major public health problem, particularly for older people, and creates an important clinical and economic burden. A high mortality rate was reported in Spain during the period 2015 to 2016; 3,101 serious cases were hospitalised with a confirmed diagnosis of flu, of which 11% died (352 cases). Furthermore, financial and health costs are greatly increased by the complications of flu; people aged over 65 years represent approximately 64% of the total costs. Seasonal flu vaccination is the fundamental strategy, as demonstrated by cost-benefit and cost-effectiveness studies. A priority objective is to improve the vaccine’s immune response and the search for and inclusion of adjuvants and immunostimulants in vaccines is a major line of research. This positioning report evaluates vaccination for older people and the importance of the adjuvanted vaccine in the elderly in strengthening immunogenicity, by means of a critical review of the literature based on the best evidence available on its immunogenicity and effectiveness, and an economic assessment (AU)

Nile Red fluorescence spectrum decomposition enables rapid screening of large protein aggregates in complex biopharmaceutical formulations like influenza vaccines.

The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components.

Determination of influenza B identity and potency in quadrivalent inactivated influenza vaccines using lineage-specific monoclonal antibodies.

Co-circulation of two antigenically and genetically distinct lineages of influenza B virus, represented by prototype viruses B/Victoria/2/1987 and B/Yamagata/16/1988, has led to the development of quadrivalent influenza vaccines that contain two influenza B antigens. The inclusion of two influenza B antigens presents challenges for the production and regulation of inactivated quadrivalent vaccines, including the potential for cross-reactivity of the reagents used in identity and potency assays because of the relative close relatedness of the hemagglutinin (HA) from the two virus lineages. Monoclonal antibodies (mAbs) specific for the two lineages of influenza B HA were generated and characterized and used to set-up simple identity tests that distinguish the influenza B antigens in inactivated trivalent and quadrivalent vaccines. The lineage-specific mAbs bound well to the HA of influenza B strains included in influenza vaccines over a period of more than 10 years, suggesting that identity tests using such lineage-specific mAbs would not necessarily have to be updated with every influenza B vaccine strain change. These lineage-specific mAbs were also used in an antibody capture ELISA format to quantify HA in vaccine samples, including monovalent, trivalent, and quadrivalent vaccine samples from various manufacturers. The results demonstrated correlation with HA values determined by the traditional single radial immunodiffusion (SRID) assay. Further, the antibody-capture ELISA was able to distinguish heat-stressed vaccine from unstressed vaccine, and was similar to the SRID in quantifying the resultant loss of potency. These mAb reagents should be useful for further development of antibody-based alternative influenza B identity and potency assays.

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.

Novel variants of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in migratory waterfowl of Hongze Lake.

Wild birds are known to play a major role in the evolution, maintenance, and spread of the avian influenza viruses (AIVs). More specifically, the waterfowl are thought to be the natural reservoirs of AIVs. Here, we conducted a survey in 2015 at the Hongze Lake and characterized 11 H5N1 highly pathogenic AIVs isolated from wild waterfowls which were found to belong to clade 2.3.2.1. In contrast, the 11 variants of H5N1 viruses did not align with the three previously defined monophyletic subclades. Antigenicity analysis revealed that antigenic drift occurred in these H5N1 variants. Hence, current vaccines may fail to confer protection against the H5N1 AIV variants in poultry.

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.

Papel de los profesionales sanitarios de los ASSIRes en la vacunación antigripal en gestantes / Role of health professionals of sexual health and reproduction clinics in the influenza vaccination in pregnant women

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Sistema de autodeclaración de acontecimientos adversos y cobertura de vacunación antigripal en profesionales sanitarios en un hospital universitario de tercer nivel / Adverse events self-declaration system and influenza vaccination coverage of healthcare workers in a tertiary hospital

Introducción: Durante la campaña de vacunación antigripal 2011-2012 establecimos un sistema de autodeclaración de acontecimientos adversos (AA) en el personal sanitario (PS). El objetivo de este estudio es describir la población vacunada y analizar la cobertura de vacunación y los AA autodeclarados tras la vacunación voluntaria del PS frente a la gripe en un hospital universitario de tercer nivel en Barcelona. Métodos: Estudio observacional. Para el cálculo de la cobertura de vacunación se utilizó el registro de vacunación de profesionales sanitarios del hospital. Los AA se recogieron mediante una encuesta voluntaria, anónima y autoadministrada durante la campaña de vacunación antigripal 2011-2012, y se analizaron mediante regresión logística. Se construyó un modelo de regresión logística para determinar los factores que predisponen a declarar AA. Resultados: La campaña alcanzó una cobertura de vacunación antigripal del 30,5% (n = 1.507/4.944) del PS. De los vacunados, el 23,8% (n = 358) respondieron la encuesta de AA autodeclarados. El 52,0% (n = 186) de los que respondieron a la encuesta declaró haber presentado algún tipo de AA. De estos, el 75,3% (n = 140) refirió signos y síntomas locales tras la vacunación, el 9,7% (n = 18), signos y síntomas sistémicos, y el 15,1% (n = 28), síntomas tanto locales como sistémicos. No se declaró ningún AA grave. Ser mujer y tener menos de 35 años se asoció a declarar algún tipo de AA. Conclusiones: El sistema de autodeclaración no registró AA graves en el PS, suponiendo una oportunidad para aumentar la confianza del PS en la vacuna antigripal (AU)

Introduction: During the influenza vaccination campaign 2011-2012 we established a self-declaration system of adverse events (AEs) in healthcare workers (HCW). The aim of this study is to describe the vaccinated population and analyse vaccination coverage and self-declared AEs after the voluntary flu vaccination in a university hospital in Barcelona. Methods: Observational study. We used the HCW immunization record to calculate the vaccination coverage. We collected AEs using a voluntary, anonymous, self-administered survey during the 2011-2012 flu vaccination campaign. We performed a logistic regression model to determine the associated factors to declare AEs. Results: The influenza vaccination coverage in HCW was 30.5% (n = 1,507/4,944). We received completed surveys from 358 vaccinated HCW (23.8% of all vaccinated). We registered AEs in 186 respondents to the survey (52.0% of all respondents). Of these, 75.3% (n = 140) reported local symptoms after the flu vaccination, 9.7% (n = 18) reported systemic symptoms and 15.1% (n = 28) both local and systemic symptoms. No serious AEs were self-reported. Female sex and aged under 35 were both factors associated with declaring AEs. Conclusions: Our self-reporting system did not register serious AEs in HCW, resulting in an opportunity to improve HCW trust in flu vaccination (AU)

New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines.

Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.

Investigation into alternative testing methodologies for characterization of influenza virus vaccine.

The objective of this study was to explore various testing methodologies suitable for characterizing sedimented or agglomerated material. To model this, bioCSL's split influenza virus vaccine, Fluvax® was utilized. The investigation was conducted on 5 dispensed lots of commercially manufactured vaccine, formulated for the 2013 Southern Hemisphere season. Vaccine syringes were initially inspected by visual tests; the material was then aseptically pooled for characterization assessment by microscopy and several agglomeration assays. All syringes passed bioCSL's description test where any fine or large sized particles of sediment observed in the vaccine were resuspended upon shaking; inverted light microscopy verified that the sediment morphology was consistent with influenza vaccine. Electron microscopic examination of pooled vaccine material demonstrated the presence of typical influenza structures including split virus, virosomes, whole virus particles and agglomerates. An optical density turbidity assay revealed relatively high protein recoveries in the vaccine supernatant post-centrifugation treatment, thus indicative of a well-dispersed vaccine formulation. This was corroborated by particle sizing analysis using dynamic light scattering which generated reproducible volume particle size distributions of a polydisperse nature. Ultraviolet-visible absorbance profiles further confirmed the presence of some agglomerated material. Data from all methods demonstrated consistent results between all batches of vaccine. Therefore, this investigation revealed the suitability and usefulness of the various methodologies in characterizing the appearance of agglomerated vaccine material. It is suggested that such methods may be applicable and beneficial for the development of a wider spectrum of heterogeneous and agglomerated formulations to provide safe, efficacious and superior quality biopharmaceutical products.

A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages.

A cell culture-based vaccine production system is preferred for the large-scale production of influenza vaccines and has advantages for generating vaccines against highly pathogenic influenza A viruses. Vero cells have been widely used in human vaccine manufacturing, and the safety of these cells has been well demonstrated. However, the most commonly used influenza-vaccine donor virus, A/Puerto Rico/8/1934 (PR8) virus, does not grow efficiently in Vero cells. Therefore, we adapted the PR8 virus to Vero cells by continuous passaging, and a high-growth strain was obtained after 20 passages. Sequence analysis and virological assays of the adapted strain revealed that mutations in four viral internal genes (NP, PB1, PA and NS1) were sufficient for adaptation. The recombinant virus harboring these mutations (PR8-4mut) displayed accelerated viral transport into the nucleus and increased RNP activity. Importantly, the PR8-4mut could serve as a backbone donor virus to support the growth of the H7N1, H9N2 and H5N1 avian viruses and the H1N1 and H3N2 human viruses in Vero cells without changing its pathogenicity in either chicken embryos or mice. Thus, our work describes the generation of a Vero-adapted, high-yield PR8-4mut virus that may serve as a promising candidate for an influenza-vaccine donor virus.