Professional Documents
Culture Documents
Vaccinegate:
Study on the chemical composition profile
of Infanrix Hexa
First revision December 16, 2018
● chemical contamination from the manufacturing process or cross-contamination with other manufacturing lines;
● chemical toxins;
● bacterial peptide toxins;
● insoluble and indigestible macromolecule that reacts to the protein assay, but cannot be recognized by any protein databases.
● Protein antigens of diphtheria toxoids, tetanus, pertussis, hepatitis B, haemophylus influenzae B, Poliomyelitis 1-2-3;
● Formaldehyde and glutaraldehyde, phenoxyethanol, antibiotic residues indicated in the composition;
reactions 6 and these too put a safety issue that needs to be further clarified with the regulatory bodies.
Coming back to the two basic principles that have been our topic on this analysis path, we reaffirm what we have said in the recent interview on
the scientific journal Nature: we are inquiring the vaccines efficacy and safety and we can't quite understand how it is possible to claim that this
vaccine is even able to generate the 6 protective antibodies - reason why it is designed for - and furthermore to understand how this cluster made
of 6 neurotoxic antigens bound together can be claimed as not toxic for newborns.
Infanrix Hexa hexavalent, as for the method we have commissioned, casts major doubts on both its effectiveness and on its safety…
One thing is for sure: we will not stop to proceed.
First revision December 16, 2018
1
Lett Appl Microbiol. 2015 Feb;60(2):174-80. doi: 10.1111/lam.12355 - https://www.ncbi.nlm.nih.gov/pubmed/25376111
2
Fuchs F., Biochimie. 2002 Nov;84(11):1173-9 - https://www.ncbi.nlm.nih.gov/pubmed/12595146
3
http://www.camera.it/leg17/491?idLegislatura=17&categoria=022bis&tipologiaDoc=documento&numero=023&doc=pdfel
4
http://www.camera.it/leg17/436?shadow_organo_parlamentare=2588
5
https://pubchem.ncbi.nlm.nih.gov/compound/formaldehyde
6
https://www.who.int/biologicals/vaccines/diphtheria/en/
7
https://www.who.int/ith/vaccines/tetanus/en/
8
http://www.who.int/biologicals/vaccines/pertussis/en/
9
https://www.who.int/biologicals/areas/vaccines/haemophilus/haemophilus_influenzae_typeb_Hib/en/
10
h
ttps://www.sciencedirect.com/topics/neuroscience/polymyxin
First revision December 16, 2018
Based on the calibration line, a protein concentration of 1.099 mg/mL was detected.
2.4 - 4th analysis: Digestion as it is at 57 ° C
After Bradford's assay, 20 μL of the raw sample has been treated with 80 μL of Trypsin. A 1 mg / mL hemoglobin control has been prepared and
treated as the sample. They have been left in thermoblock at 37 ° C for 4 hours and then at 57 ° C for 30 minutes. The sample and the control have
First revision December 16, 2018
then been subjected to centrifugation and the supernatant has been taken and placed in vial for analysis.
11
In order to process the data thus obtained, the Mascot database has been initially used but nothing has been found. Therefore, the GMP has
been used but also in this case no protein sequences have been detected. By the DeNovo research, the following peptide sequences that do not
meet the trypsin cutting criteria and therefore potentially belong to free peptides have been identified. Below is the detected sequences list:
YLSA YLSA SLGS HNLPFT
SNCGYY VFHRF
12
These sequences have been filed into the MS-BLAST search engine obtaining the characterizations reported in Table 1. As can be seen, they
have been potentially attributed by structural similarity to proteins of the bacterial world. The proteins relating to the antigens present in the
vaccine have not been detected. This may be due to its extensive structural modification, introduced by formaldehyde and glutaraldehyde. In fact,
the database research has been carried out Without considering the m/z variation introduced by the above-mentioned compounds
It is important to verify whether these changes have led to cross-linked macromolecular complexes shaping. In this regard, we will ask for further
13
analysis using MALDI-TOF-MS technology widely acknowledged in clinical practice for the study of high weight macromolecules,
■ Homeodomain-like DNA binding domain-containing transcription factor ■ Phycomyces blakesleeanus NRRL 1555(-) 110
■ glycosyl hydrolase family 3 N-terminal domain protein ■ Firrmicutes bacterium CAG:56 108
■ PREDICTED: zinc finger CCCH domain-containing protein 69-like ■ Pyrus x bretschneideri 108
11
h
ttp://www.matrixscience.com/help/seq_db_setup_db_gui.html
12
h ttp://genetics.bwh.harvard.edu/msblast/
13
h ttps://it.wikipedia.org/wiki/MALDI
First revision December 16, 2018
Unknown compounds
Known compounds
In Batch # 1 we have 65 signals of which only 35% are known
It has not been possible to carry out the analyzes on different batches because since
all the Infanrix Hexa we have purchased for more than a year on the national
territory, in different regions and in different periods, belong to batch A21CD072D.
NOTES FOR UNDERSTANDING: this is a first level analysis, that is an identification based on molecular weight. If the result is
univocal, (ie at a measured molecular weight only one compound is associated as a structure) it is more likely the right one, but
absolute certainty is not possible in this phase. As you will notice, as far as a certain number of compounds is concerned,
different substances correspond to a particular molecular weight.
3.1. Infanrix Hexa Vaccine - Batch # 1 (A21CD072D)
65 signals were detected, among which only the 3
5% gave a potential classification (Table 2).
It must be specified that compound identity is not certain and it should be confirmed by a second level screening carried out with certified
analytical standards.
In fact, during screening level, the device measures a particular data by its accurate molecular weight (measurement error <10 ppm). The empirical
formula is calculated on the basis of these measures. Some formulas might correspond to several compounds having the same molecular weight
but different chemical identity.
NOTES FOR UNDERSTANDING: in essence, the certain data is that we have 65 chemically different substances among which
only 35% is known.
Molecules potentially belonging to toxin category have been researched. They have been suggested on the basis of accurate m / z mode research
(error <10 ppm) using the toxic compounds database in the Metlin search engine. Table 3 shows the candidates obtained.
First revision December 16, 2018
It is emphasized that different detected compounds have a candidate empirical formula containing sulfur compounds or sulfur in the form of
various functional groups. Furthermore, the presence of formic acid in the form of sodium salt and a polymer deriving from contaminations of
14
Poly Ethylene Glycol (PEG) with an average molecular weight equal to 1340 Da have been detected.
5. Final considerations
Most of the contaminants and impurities detected were not characterized using the metabolic and protein reference databases (KEGG, NCBI-Prot e
SwissProt). 8-9
There is a critical issue in the contamination of various compounds potentially or definitely harmful to human health.
In short, the first questions we asked ourselves, and the relative answers obtained, are the following:
1. Are the chemical substances listed in the data sheet present? in part
Next analysis
1. First of all, it is necessary to identify with certainty the most interesting probable compounds
3. Finally to determine the structure of the macromolecule constituted by the set of antigens
6. Future research developments
Confirmation and identity analysis will be performed using the "Tandem Mass Spectrometry (MS / MS)" technique associated with the aid of
certified analytical standards. The analysis will be performed in compliance with the European directives (EU directive 2002/657 / EC) useful for the
identification of compounds.
In particular, the investigation will have the objective of confirming those substances whose toxicity and allergenicity is known and the 7 toxins
identified will be an element of careful study.
7. Description of the SANIST technology
15 16
The innovative internationally renowned SANIST platform, through publications in peer-reviewed scientific journals - - was used to perform a
first identification screening on the vaccines of interest.
14
h
ttps://www.sciencedirect.com/topics/materials-science/polyethylene-glycol
15
Albini A. et al., Rapid Commun Mass Spectrum. 2015 Oct 15;29(19):1703-10. doi: 20.1002/rcm.7270. (https://onlinelibrary.wiley.com/doi/full/10.1002/rcm.7270)
16
Cristoni S. et al., J Mass Spectrom. 2017 Jan;52(1):16-21. doi:10.1002/jms.3895. (https://www.ncbi.nlm.nih.gov/pubmed/27776380)
First revision December 16, 2018
17
Kanehisa M. et al., Nucleic Acids Res. 2017 Jan 4;45(D1):D353-D361. doi:10.1093/nar/gkw1092. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210567/)
18
Cristoni S. et al., Expert Rev Proteomics. 2004 Dec; 1(4):469-83. (https://www.ncbi.nlm.nih.gov/pubmed/15966842)
First revision December 16, 2018
Table 2 - Batch #1 (A21CD072D)
■ Tungsten carbide ■Inorganic carbide industrially used to synthesize cemented carbides.
■ Lichenin (altri 19 possibili candidati) ■ Glucan. Study to use glucans as vaccine adjuvants
■ Justicidin B ■ Lignan
■ Deamino-alpha-keto- ■ -
demethylphosphinothricin
■ Cassythine ■ Alkaloid
■ (6-alpha-D-glucosaminyl)-1D-myo ■ A derivative of a D-glucosaminide and a monosaccharide.
inositol
■ Amoxicillin ■ Antibiotic
■ Cephalexin monohydrate ■ Antibiotic that decreases the effectiveness of vaccines
■ Cefroxadine ■ Cephalosporin antibiotic
■ CGP 28-392 ■ Aromatic ether
■ 2-amino-5-chloromuconate-6 ■ Semialdehyde
-semialdehyde
■ 1-Palmitoyl-2- ■ 1,2-diacil-sn-glicero-3-fosfocolina
(5-hydroxy-8-oxo-6-octenoyl)-sn-
glycero-3-phosphatidylcholine
First revision December 16, 2018
■ Hepta-2,3,4,5,6-pentaenenitrile C7H3N
■ (Methanesulfonyl)(dioxo)-lambda~5~-azane
CH3NO4S
■ Oxaziridine-2-sulfonic acid
■ 3,4-Dihydro-3-thioxo-1,2,4-triazin-5(2H)-one
■ 5-Thioxo-4,5-dihydro-1,2,4-triazin-3(2H)-one
■ 1,2,4-oxadiazole-3-carbothioamide
■ 1,2,3-Thiadiazole-4-carboxamide C3H3N3OS
■ 5-amino-1,3,4-thiadiazole-2-carbaldehyde
■ 1,1-Difluoro-1-isocyanatoethane
■ 1,1-Difluoro-2-isocyanatoethane
■ 3-(methylsulfonyl)-1H-1,2,4-triazole
■ 1H-Imidazole-2-sulfonamide
■ N-(4,5-Dihydro-1,3-thiazol-2-yl)nitramide
C3H3F2NO
■ 1H-Imidazole-5-sulfonamide
■ 1H-Pyrazole-4-sulfonamide
■ Undeca-2,4,6,8,10-pentaynenitrile
■ 2-Aminobenzenethiol
■ 4-Methyl-5-vinylthiazole
■ 2-Pyridinemethanethiol
■ 2-Isopropenylthiazole
C6H7NS
■ 5,6-Dihydro-4H-cyclopenta[d][1,3]thiazole
■ 4-Aminothiophenol 3-Pyridinemethanethiol
■ Pyridine, 2-(methylthio)
■ Pyridine, 3-(methylthio)-
■ Phenthiazamine C9H8N2S
■ 4-Chloro-N-hydroxybenzene-1-sulfonamide C6H6ClNO3S