Attachment E: TGA influenza seasonal trivalent influenza vaccine laboratory investigation program
8 October 2010
Following reports of increased fever, the TGA immediately re-examined batch release data, as well as the results from original compendia testing, carried out by the Office of Laboratories and Scientific Services (OLSS) for batches of Fluvax that had been used in Western Australia.
The TGA planned an investigation that would urgently test for the presence of lipopolysaccharide (LPS) from gram-negative bacteria, which is associated with febrile events, by measuring the endotoxin content of the vaccines. Furthermore, the potency of the batches was tested to check general quality as well as to ensure that the vaccine did not contain an unusually high content of antigen that may trigger a febrile event.
Further to these tests, the TGA has been conducting an investigation and research program to determine whether there is a biological or physicochemical basis to the vaccine product itself that may be contributing to the apparent increase in febrile events related to the 2010 influenza vaccine program.
To assist with this testing, the TGA has sought advice from a panel of experts, chaired by Professor Peter Doherty, and has had discussions with the US Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), as well as the UK's National Institute for Biological Standards and Control (NIBSC), which have offered advice and assistance with the testing program.
Haemagglutinin is one of the main antigenic determinants of influenza viruses and its content in the vaccine is a principal measure of the quality and efficacy of the vaccine. Furthermore, it is understood that haemagglutinin, amongst other proteins, plays a major role in the pyrogenicity of the influenza vaccine. Therefore the content of haemagglutinin antigen was determined in field samples from WA to check the quality and to determine if there were unusually high amounts of the antigen that may contribute to increased pyrogenicity.
The content of haemagglutinin antigen was determined by immunodiffusion techniques using the Single radial immunodiffusion (SRID) assay. Tests were carried out on field samples of Fluvax from WA. Previous test results from tests carried out by the OLSS at the time of batch release were re-examined for batches of Fluvax, Vaxigrip and Influvac.
All field and retention samples passed the monograph requirements and no unusual values for the content of haemagglutinin antigen were noted.
Lipopolysaccharides (LPS) are found in the outer membrane of various gram-negative bacteria and are an important endotoxin that can cause fever. Endotoxins are present during the manufacture of the influenza vaccines and processes are in place to eliminate and test for the presence of these chemicals. Therefore, the content of endotoxin was measured in the final product by the TGA to check that it was not contributing to the increased pyrogenicity. [Endotoxin (Wikipedia)]
Bacterial endotoxin of gram-negative bacterial origin was quantified using amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus), based on the gel-clot technique whereby the lysate clots in the presence of endotoxins. Tests were carried out on field samples of Fluvax from WA. Previous test results from tests carried out by the OLSS at the time of batch release were re-examined for batches of Fluvax, Vaxigrip and Influvac.
All field samples tested passed the monograph requirements, which is 100EU/dose. All batch release testing passed monograph requirements. Endotoxin content was mostly <1.2EU/mL with the highest sample showing <6EU/mL. Given that the monograph requirement is 100EU/dose, the results indicate that the content of endotoxin is extremely low, with the highest being 1.5EU/dose in children.
There are a number of processing agents used during the manufacture of the influenza vaccines that are present in trace amounts in the final product. Furthermore, the same production methodology is applied to the same vaccine product from different years. Therefore, chemical profiles of different batches within a given year and between years can be used to determine whether any general abnormalities or contaminants are present. Any significant differences could lead to further inquiries to assist in determining a cause of increase pyrogenicity.
Chromatographic profiling (high performance liquid chromatograph fitted with a photodiode array UV/Vis detector) was used to compare the profile of several batches of the 2010 Fluvax with that of the 2009 Fluvax product. Additionally, two other flu vaccine products (Vaxigrip 2009 and 2010 and Influvac 2009 and 2010) were run for additional comparison. Each of the samples was run without dilution under identical chromatographic conditions in order to observe significant differences, if any, between the profiles of individual samples.
No contaminants or significant differences were noted between batches within a given year or between years for each of the vaccine products. These results did not provide any evidence to pursue other lines of inquiry related to pyrogenicity.
It is understood that proteins such as haemagglutinin, with a molecular weight (MW) of 77,000 Da, and neuraminidase (60,000 Da) can play a major role in the pyrogenicity of the influenza virus. These are surface membrane glycoproteins that exist in oligomeric forms with the haemagglutinin found as a trimer (230,000 Da) and the neuraminidase as a tetramer 240,000 Da. Monomeric haemagglutinin is itself a heterodimer composed of two subunits – around 50,000 and 30,000 Da. Other protein constituents include matrix proteins (28,000 and 12,000 Da) and nucleoprotein (55,000 Da). Along with several minor components origination from the membrane lipid matrix, these proteins are found at varying levels in vaccine preparations.
Studies reported in the literature suggest that a vaccine manufactured by the same method by the same manufacturer should have a particular chromatographic profile that can be used as a signature for that particular vaccine product. Therefore, any significant changes to the profile for batches of vaccine from the same manufacturer may suggest that the properties of the vaccine are different and may indicate where further investigation is required. Further, differences in the oligomeric forms of the proteins may in some way be related to their potential to be pyrogenic. For example, if there is an increase in the portion of monomers of haemagglutinin, does this influence the pyrogenicity of the vaccine? More generally, chromatographic profiling of the protein can provides evidence of a 'point of difference' for the purposes of further investigation.
A suitable size exclusion high performance liquid chromatography (SE-HPLC) technique was adopted from a study by Garcia-Canas et al (2010). Samples from 2010 and 2009 vaccine batches were injected in triplicate (for Fluvax samples) and duplicate (for Vaxigrip and Influvac samples). Molecular weight reference standard preparation for SE-HPLC analysis (BioRad) was injected for molecular size estimation.
Mass spectrometry techniques are being used to assess and gain a greater understanding of the content, form and distribution of each protein within the different vaccines.
Protein characterisation using size exclusion HPLC has shown different characteristic profiles for the different vaccines, which is expected given differences in the manufacture of each product. For each of the vaccines, there appears to be greater protein aggregation in the 2009 samples compared with 2010 samples, which may reflect increased protein aggregation occurring during the life of the vaccine. Nevertheless, the profiles suggested that the content of one of the proteins in the 2010 seasonal influenza vaccine may be higher than in previous years (see Fig 1 below).
Initial results from mass spectrometry studies indicate that the neuraminidase content of the H1N1 component of this year's seasonal influenza vaccine is higher than previous influenza vaccines.
The influenza vaccines are either split virion (Fluvax and Vaxigrip) or subunit (Influvac) products. The pharmacopoeial requirement for split virion inactivated influenza vaccine requires that the vaccine consists predominantly of disrupted virus particles. Tests must be carried out by the manufacturer to demonstrate that the virus is inactivated. Therefore the presence of whole virus particles and live virus in the finished product was determined by the TGA to check whether this may contribute to increased pyrogenicity.
Transmission electron microscopy and cell culture assays were carried out by the OLSS at the Department of Anatomical Pathology, and the Virology Department, at the Institute of Clinical Pathology and Medical Research, Westmead Hospital. Samples from the following vaccines were examined: Fluvax, Fluxax Junior, Panvax, Panvax Junior, Vaxigrip, Intanza and Influvac. Influenza virus infectivity for all the vaccines was examined in Madin-Darby canine kidney (MDCK) cells. All samples were looked at directly by electron microscopy (EM) and samples of vaccine were also concentrated by ultracentifugation and the sensitivity of Electron microscopy was further enhanced with the use of polylysine coated grids.
No intact viral particles or live virus was detected in the final products. Moreover, no intact viral particles were detected in ultracentrifuged samples or with the use of more sensitive polylysine coated grids.
It is acknowledged that the final product is a very dilute form of the vaccine and it was unlikely that whole particles would be found by direct EM. However, whole virus particles were also not detected using more concentrated samples of vaccine and more sensitive EM techniques. Nevertheless, a significant presence of intact virus particles would have been of concern with regard to potential pyrogenicity from whole virus particles.
Particular cytokines (IL-1, IL-6, TNFα and IFNα) can be associated with febrile events. Therefore, the capacity of the vaccines to induce cytokine expression is being investigated, using in-vitro pyrogenicity studies, to determine if there are differences between vaccines and differences with vaccines from different years.
Samples of vaccine were tested in vitro for their ability to stimulate transfected human peripheral blood mononuclear cells (PBMCs). Supernatants from the cells (collected from one individual) were measured for the expression of cytokines TNF-α and IFN-α using ELISA assays. The experiment was conducted in the laboratory of Prof. Bryan Williams, Faculty of Medicine, Nursing and Health Sciences at the Monash Institute of Medical Research. The assay is routinely used in this laboratory to investigate the ability of RNA molecules to stimulate the innate immune response, and was used to test whether the different vaccines had differential stimulation of this response, with TNF-α particularly linked to pyrogenicity. Appropriate controls were included to examine the impact of the vaccine formulations on the cell transfection process.
Samples of vaccine were sent to NIBSC to investigate TNF-α stimulation in a monocyte activation test using blood from four adult donors.
The different vaccines appear to have a different capacity to inhibit the assay and any results must be interpreted with great caution. In particular, Vaxigrip seem to totally inhibit the assay, with Influvac appearing to have a greater inhibitory capacity compared with Fluvax or Panvax. Nevertheless, Fluvax samples did seem to stimulate relatively greater levels of TNFα expression than Panvax, Influvac and Vaxigrip, but the 2010 Fluvax samples were no more stimulatory than the 2009 Fluvax samples. No differential pattern of IFNα expression between the vaccine samples was observed, except the lack of expression induced by Vaxigrip samples consistent with its apparent inhibition of the assay.
Results showed that there was a greater percentage of TNF-α positive lymphocytes following stimulation with Fluvax 2010 and Fluvax 2009 compared with stimulation with Influvac 2010 and 2009. However, there was no difference in the percentage of TNF-α positive lymphocytes following stimulation by Fluvax 2010 compared with Fluvax 2009.
The TGA has been advised of results from studies in other laboratories that indicate IFNα and IL-6 expression appears to be greater with Fluvax compared to Panvax and Vaxigrip, but there is not a clear difference in the expression between Fluvax 2009 and 2010. Results from studies by CSL hint that Fluvax 2010 stimulates greater expression of IL-1, IL-6 and TNF-α than Fluvax 2009. However, the expression levels are extremely low and difficult to interpret.
The possibility has been raised that the presence of RNA, particularly in the Fluvax, may be causing pyrogenicity. Therefore, the OLSS is seeking to determine whether RNA is present in the vaccine.
RNA quantification using an Agilant Bioassay Kit – Bioanalyser.
Initial tests have not detected the presence of RNA in Fluvax, however more sensitive tests are planned to try to detect and quantify any presence of RNA.
Given the reports of febrile events, the capacity of the vaccines to produce pyrogenicity in animal models is being investigated.
Samples of Fluvax, Fluxax Junior, Panvax, Panvax Junior, Vaxigrip, and Influvac are being tested in standard ferret and rabbit pyrogenicity models, with associated weight loss and cytokine measurements.
Testing by CSL indicates that Fluvax 2010 passes pharmacopoeial requirements for rabbit pyrogenicity tests and there was no difference between 2009 and 2010 Fluvax. The company concluded that none of the formulations tested induced pyrogenic reactions in the rabbits of a magnitude that would indicate that a pyrogen may be the causative agent of the febrile responses in children under 5.
Analysis of results from pyrogenicity studies in ferrets suggests that there may be significant differences in changes to body temperature following inoculation with Fluvax compared to other vaccines. These studies are on-going in an effort to obtain more detailed information.
|SampleName||Peak Name||Retention Time (min)||Area||%Area||Height|
|1||Fluvax 2009 B@0906-25401||Peak 1||11.801||12486994||38.11||163001|
|2||Fluvax 2009 B@0906-25401||Peak 2||13.771||11246779||34.33||149389|
|3||Fluvax 2009 B@0906-25401||Peak 3||15.726||3054682||9.32||29450|
|4||Fluvax 2009 B@0906-25401||Peak 4||18.494||4196833||12.81||32790|
|5||Fluvax 2009 B@0906-25401||Peak 5||22.876||439238||1.34||6737|
|6||Fluvax 2009 B@0906-25401||Peak 6||25.258||417341||1.27||5014|
|7||Fluvax 2009 B@0906-25401||26.428||248397||0.76||4378|
|8||Fluvax 2009 B@0906-25401||27.376||367129||1.12||3858|
|9||Fluvax 2009 B@0906-25401||29.017||304725||0.93||2296|
|SampleName||Peak Name||Retention Time (min)||Area||%Area||Height|
|1||Fluvax 2010 B@0906-27201||Peak 1||11.867||11763197||27.86||131581|
|2||Fluvax 2010 B@0906-27201||Peak 2||13.774||17690160||41.90||240898|
|3||Fluvax 2010 B@0906-27201||Peak 3||15.782||5203542||12.32||47171|
|4||Fluvax 2010 B@0906-27201||Peak 4||18.175||5417385||12.83||45498|
|5||Fluvax 2010 B@0906-27201||Peak 5||22.583||882700||2.09||9371|
|6||Fluvax 2010 B@0906-27201||Peak 6||25.431||318305||0.75||4247|
|7||Fluvax 2010 B@0906-27201||26.446||308637||0.73||4321|
|8||Fluvax 2010 B@0906-27201||27.226||640170||1.52||3751|
Content last updated: Friday, 8 October 2010
Content last reviewed: Friday, 8 October 2010
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