Warsaw 8th of January 2019 In- depth investigation of vaccines composition

1. Warsaw 8th of January 2019 In-depthinvestigation of vaccinescomposition Dr. Loretta Bolgan

2. The ItalianParliamentaryCommission of inquiry on the cases of death in the armypersonnel With the resolution of 30 June 2015, published in the Official Journal no. 160 of the 13 July 2015, the Chamber of Deputiesestablished the «ParliamentaryCommission of Inquiry on the cases of death and seriousillnessesthathaveaffectedItalianpersonnelemployed in militarymissionsabroad, in shootingranges and ammunitionstoragesites, in connection exposure to particularchemical, toxic and radiologicalfactors with a possiblepathogeniceffect and by the administration of vaccines, with particularattention to the effects of the use of projectiles with depleteduranium and the dispersion of heavymineralnanoparticles in the environment, produced by explosions of war material and possibleinteractions»

3. Tasks of the ParliamentaryCommission among the tasks of the Commissiontherewas the investigation d) on the components of the vaccinesgiven to militarypersonnel, regardless of the subsequent use of the samepersonnel; e) on the methods of administration of vaccines to militarypersonnel, aswellason monitoring the immune status of the observedsubjects, takinginto account in particular the results of the projectcalled "Study on the genotoxic impact in militaryunits" (SIGNUM); (GU n.160 of 13-7-2015)

4. The study of prof. Franco Nobile on the Folgore Brigade SENATORIAL COMMISSION OF INQUIRY ON DEPLETED URANIUM AND OTHER CAUSES OF HARMFULNESS FOR OUR ARMY PERSONNEL- AUDITION PROF. NOBILE (2010) the testswereconducted on a group of soldierswhohadnevergone on a mission, and on a group of soldiersreturning from severalmissions. Thesetestsshowed a frequentimmunologicalalteration with a reduction of the ratio in T lymphocytes (CD3+) between CD4+ and CD8+ and in analogy with whathappens in AIDS and in terminal cancerpatientswasstrongly suggestive of immunodepression, linked to the vaccinationpractice, whichcould in turn be the basis of autoimmune diseasessuchas autoimmune thyroiditis, multiple sclerosis, nodularerythema, lupus, rheumatoidarthritis, diabetes, opticneuritis and, according to some researchers, leukemia and lymphomas

5. SIGNUM PROJECT • Study on the Genotoxic Impact in MilitaryUnits; final report on 17 January 2011 • the studywasconceived on the basis of a voluntarymembership of the military (981) belonging to a nationalrotationcontingentdestined for the Iraqi Op. Te. • The overall target of the projectwas to evaluate the exposure to tumorriskfactorsduring the missionperiod and anyearlyindicators of potentialbiologicaldamageassociated with them. RESULTS: «The indicationthatemerges from theseresultsisthat: the use of vaccine trials with more than 5 differentvaccinesin rare subjects with OGG1 / GSTM1 riskgenotype and the use of thesesubjects in patrollingactivitieswouldnot be appropriate. »

6. FINAL REPORT OF THE PARLIAMENTARY COMMISSION (On. Ivan Catalano) February 2018 In November 2017, the Commissionreceived the documentationrequested from AIFA in spring 2016, concerningtechnicalspecifications, safetystudies and vaccine composition, including sub-thresholdelements. RESULTS The adoption of practicessuchas a calendar of combinedvaccinations with multivalentvaccines can, in itself, represent a healthrisk in relation to atleastthreeaspects: ● the cumulative amountof the variouscomponents of the vaccinesexceedsthe permittedlimitfor the marketing authorization of the individual vaccine; ● the hypersensitivityindicated in the registrationdossiers and technical reports of the vaccines, confirm the need for pre-vaccinaltests; ● the adversereactionsreported in the registrationdossiers and technical reports of the vaccinesconfirm the need for a personalizedriskassessmenton vaccine prophylaxis and the need for long-termperiodicmonitoringof eachindividualvaccinee. The Commission once againconfirms the conclusionsalreadyhighlighted by the SIGNUM Project, aswellas by the work of Prof. Nobile on the Folgore Brigade - thatis, the neednot to simultaneouslyadminister more than 5 monovalent single-dose vaccines on the military.

7. Definition of vaccine A vaccine is a biological preparation thatimprovesimmunity to a particular disease. A vaccine typicallycontains an agent thatresembles a disease-causingmicroorganism, and is often made from weakened or killedforms of the microbe, itstoxins or one of itssurfaceproteins. The agent stimulates the body's immune system to recognize the agent asforeign, destroyit, and "remember" it, so that the immune system can more easilyrecognize and destroyany of thesemicroorganismsthatitlaterencounters. www.who.int/topics/vaccines/en/

9. Marketing Authorisation (AIC) Itis the actthatallows a pharmaceutical company to market a medicine, specialty or generic, produced in an industrial way. There are currentlythreetypes of marketing authorization in the EU: National, Europeanaccording to a centralized procedure, European for mutualrecognition (or decentralized) The marketing authorizationisissuedfollowing the assessment of the risk-benefit ratio of the vaccine on the basis of a dossier in whichall the data collectedduringproductdevelopment and clinicalstudies are documented. The assessmentcovers the properties of the product, suchasquality, safety and efficacy. The regulatoryagenciesresponsible for the evaluation of the dossier must ensurethat the manufacturermeetsall the requirements of the rules of goodworkingpractice(GMP: Good Manufacturing Practicerefers to a system of rulesaimedatensuringthat the drugs are produced in a coherent and controlled way to guarantee precise qualitystandards and minimize the risksassociated with production according to detailedprotocols, aswellassystemsthatdocument the correctapplication of the procedures in each single production phase.)

10. The batch release Once each batch of vaccine hasbeenobtained, the quality control must be performedbefore release; thisassessmentiscarried out both by the manufacturer and the European network of authorized control laboratories (OMCL Network); the activities of theselaboratories are coordinated by the EuropeanDirectorate for the Quality of Medicines & HealthCare (EDQM), whichcoordinates the development of qualitystandardsknownasmonographs of the EuropeanPharmacopoeia; once adopted, thesemonographsbecomemandatoryqualitystandards in allmemberstates of the Council of Europe and coordinates the work of the Batch Release Official Control Authority for the issue of the lot.

11. Vaccine components The vaccinesconsist of: antigens adjuvants preservatives excipients They can containimpurities and contaminations (chemical, biological and elementary)

12. Types of antigens

13. Type of adjuvants Vaccine. 2015 Jun 8;33 Suppl 2:B14-20. New generation adjuvants--from empiricism to rational design. O'Hagan DT1, Fox CB2.

14. Impurities Impurity: any component present in the substance or pharmaceuticalproductwhich is not the desired end product (activesubstance and excipientsstated in the composition). Itcould be eitherprocess-related or product -related. • Process-relatedimpurities: impuritiesderived from the manufacturing process. They can be derived from cellularsubstrates (eghostcellproteins, hostcell DNA), cell culture (eginducers, antibiotics, or medium components), or downstream processing (e.g., reagents or releasedsubstances) from the columns of purification). • Product-relatedimpurities: molecularvariants of the desiredproduct (egprecursors, degradationproductsresulting from manufacturing and / or storage) that do nothavepropertiescomparable to those of the desiredproduct with regard to activity, efficacy and safety.

15. Contaminants Contaminants in a product include alladventitiouslyintroducedmaterialsnotintended to be part of the manufacturing process, suchaschemical and biochemicalmaterials (e.g., microbialproteases), and/or microbialspecies. Contaminantsshould be strictlyavoided and/or suitablycontrolled with appropriate in-processacceptancecriteria or actionlimits for drugsubstance or drugproductspecifications. For the special case of adventitiousviral or mycoplasmacontamination, the concept of actionlimitsisnotapplicable. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002824.pdf

16. Preliminary screening of the components Analysis of the geneticmaterial The studycommissioned by Corvelvawasbased on twotypes of analysis: 1. Test of presence of nucleicacids(DNA / RNA) of human and animalorigin and of microorganisms (viruses, bacteria) using the Next Generation Sequencingmethod, whichallowed to quantify in a highlyspecific and accurate the sequence of the geneticmaterialcontained in the vaccinesexamined 2. Verification of the correspondence of the genomesequences of live attenuated or inactivatedbacteria and virusespresent in the vaccines (presence of geneticvariants)

17. Next Generation Sequencing, alsoknownasdeepsequencing, generates a single sequence from each DNA fragment, or cDNA, present in a sample. The downstream bioinformaticanalysisallows the differentiationbetween the origin of the sequencefragments, for example human, bacterialspecies or a particular virus. Thismeansthatmixed biological samples can be easilysolved with thistechnology, whichhasnowentered the routine of genomicresearch and diagnostics. Moreover, from NGS data it is possible to reconstruct the entiresequence of viral DNA and RNA genomes and bacterialgenomespresent in the sample and compare it with the referencegenomespresent in public databases.

18. Analysis of chemical and proteiccomponents The examination of military vaccine registrationdossiersrevealed the presence of contaminations and impurities of chemical and proteic nature, whichrequiredfurtheranalyticalanalysis. Itwasthereforenecessary to find a technologyable to analyze a wide spectrum of molecules of chemical, metabolic and proteicorigin to evaluate the quality of the productsobtained. The internationallyrenownedSANIST platformhasbeenused to perform a first identification screening on the vaccines of interest

19. SANIST technology It’scomposed by: • a kit for the extractionof analytes (the unknownsubstances to be determined) • The LC-SACI / ESI-MS analysissystemthatallows to reduce the chemicalnoise of mass spectrometers and obtain a betterdetection of instrumentalsignals. • The SANIST data processing systemconsists of a localbioinformatics and network platformcapable of processing data usingdedicateddatabases and customizedalgorithms. It is specifiedthat, during the screening phase, the identification is made in the context of scientificresearch and throughresearch in officialbanks (KEGG, NCBI-Prot and SwissProt) without the aid of certifiedanalyticalstandards. It is thereforenecessary to perform a secondlevelanalysis with certifiedanalyticalstandards to confirmtheiridentity.

20. Priorix tetra The measles virus and the mumps virus is obtained by propagation in chickembryotissuecultures; the rubella virus and varicella virus are propagated in MRC-5 human diploidcells. The MRC-5 cell line was derived from normal lung tissue of a 14-week-old male fetus in 1966; the cells are capable of 42 to 46 populationdoublingsbefore the onset of senescence. https://ca.gsk.com/media/591336/priorix-tetra.pdf

21. Infanrixhexa

22. Diphtheria toxoid: the diphtheria toxoid is obtained by inactivating the toxin (produced by Corynebacteriumdiphtheriae) with formaldehyde at 37 ° C in a slightly alkaline environment and then it’s adsorbed on aluminum salts (hydroxide and phosphate) Tetanus toxoid:The tetanus toxoid is obtained with the same procedure of the diphtheria toxoid (the Clostridium tetani is an obligate anaerobic bacterium and produces tetanospasmin, a neurotropic toxin that acts through the blockade of inhibitory synapses of the reflex muscle contraction) Pertussis toxoid: the components of acellular pertussis vaccine are obtained by extraction and purification of phase I cultures (primary coltures) of Bordetella pertussis (an aerobic coccobacillus, capable of producing four toxins: pertussis toxin, adenylate-cyclase toxin, dermonecrotic toxin, tracheal cytotoxin and two types of lipopolysaccharide, for the preparation of the acellular vaccine is purified and used only the pertussis toxin), followed by irreversible detoxification of pertussis toxin by treatment with glutaraldehyde and formaldehyde and formaldehyde treatment of haemagglutininfilamentosa and pertactin components; the components are then adsorbed on aluminum salts. Hepatitis B surface antigen: is produced from genetically modified Saccharomycescerevisiae cultures encoding the hepatitis B virus surface antigen gene; this antigen is purified by various chemical-physical steps and is spontaneously assembled into spherical particles of about 20 nm of diameter containing the polypeptide antigen and a phospholipidic matrix. This antigen is then adsorbed on aluminum phosphate. Poliovirus (inactivated):The Salk vaccine, or inactivated polio (IPV), is based on three wild, virulent strains of reference: Mahoney (poliovirus type 1), MEF-1 (poliovirus type 2), and Saukett (poliovirus type 3), grown in the VERO cell line: this is an immortalized cell line obtained in 1962 by the kidneys of adult African monkeys (Cercopithecus); for the production of the vaccine cells are subjected to 130-140 propagation steps, (low level of propagation), it should be noted that over 200 passages the cell line becomes carcinogenic in mice; the growth medium for the growth of the VERO line is derived from animals (and therefore must be tested for the presence of viruses and contaminating prions), while for the growth of the virus is used the Medium 199, which does not contain substances derived from animals. After being isolated and purified, live viruses are inactivated with formaldehyde. Haemophilusinfluenzae type b polysaccharide: it is prepared from the bacterial strain Hib 20,752 (invasive infections such as meningitis are mainly caused by capsulated strains, especially type b) The polysaccharide is obtained from the growth of the bacterial strain in a synthetic culture medium and after activation with cyanogen bromide and derivatization with a hydrazide-adipic spacer is coupled with the tetanus toxoid via carbamide condensation; after purification, the conjugate is adsorbed on aluminum salts and then lyophilized in the presence of lactose as a stabilizer. Conjugation with tetanus toxoid is necessary to confer antigenicity to polysaccharide as it changes the polysaccharide from T-independent antigen to T-dependent antigen. Finished product The sterile concentrates adsorbed on aluminum of DT, PT, FHA, PRN, and HBsAg and the trivalent component of IPV are mixed with a sterile solution of sodium chloride and water for injections and added with a sterile 2-phenoxyethanol solution. 2-phenoxyethanol is an antimicrobial agent and is added to the finished product because the final sterilization cannot be performed by filtration of the DTPa-HBV-IPV component and the opalescence of the suspension could mask microbial contamination. https://www.ema.europa.eu/documents/scientific-discussion/infanrix-hexa-epar-scientific-discussion_en.pdf

23. Results of chemical, proteic and metagenomicanalysis of Priorix tetra

24. Results of the DNA-seq and RNA-seqanalysiscarried out on the twobatches with the Kraken software. The presence of DNA and RNA is expressedas the number of reads and percentage of reads on the total of the readsproduced, attributed by the public databases to the variousorganisms. Results of the DNA-seq and RNA-seqanalysiscarried out on the twobatches with the Kraken software. The presence of DNA and RNA is expressedas the number of reads and percentage of reads on the total of the readsproduced, attributed by the public databases to the variousorganisms.

27. The Priorix Tetra is a vaccine with a high amount of extraneouscontaminant DNA of which 80% on average is human, coming from the MRC-5 cells; in Batch #1 there is also 4% of DNA coming from embryochickencells; the remaining 20% belongs to viruses (antigens, retroviruses, infectious and carcinogensviruses, phages) and adventitiousmicroorganismssuchasbacteria and worms. In the Priorix Tetra vaccine the human genomic DNA has high molecularweight (> 10.000bp) and the totalsequentialcoverage of the entirereference human genome (HG- 19). The EMA'sanswer to ourquestionabout the limitsimposed on residues of foreigngeneticmaterial in vaccines, confirmedthat the maximum limitranges from 10 pg to 10 ng , based on the theoreticalcalculation of the possibility of foreigngenomic DNA, derived from continuouscelllines, to cause oncogenicmutations. EMA hasnotprovidedspecificstudies on the danger of fetalresidual DNA, necessary to assess the risk of theseimpurities to human health, so thislimitremainsunknowntoday. Note: Rubellaantigen is missing in allbatchestested! (the cumulative percentage of all the adventitiousvirusesishigherthan the percentage of the mumpsantigen)

28. Analysis of the compositiondeclared in the SCP (chemical and proteic) In bothbatches are present the followingcompoundsdeclared in the summary of productcharacteristics: Amino acids, Lactose, Mannitol, Sorbitol, Water and Neomycinsulphate. Peptide fragmentsassociated with proteinsprobablyderived from the manufacturing processhavebeendetected in both production batches. WehavefoundSarcoplasmiccalcium-bindingprotein, Actin and Vimentin. Sarcoplasmiccalcium-bindingprotein is a recognizedallergen . It can be hypothesizedthatinjectedforeignproteinscould cause hypersensitivity and allergicreactions, especiallyafter vaccine recalls, butalsoautoimmunity due to theirsimilarity with human proteins. BothActin and Vimentin are of animalorigin, bovine and chickenrespectively, asimpurities from the cell culture medium

29. Analysis of metabolicfraction Itshould be notedthatthis screening studyprovides semi-quantitative data, whichranges from nanograms to microgramsas an indicative order of magnitude. Itwill be necessary to proceedusingcertifiedanalytical standard in order to obtain accurate qualitative and quantitative data. During the screening phase, instrumentsmeasure a particularsignal by its accurate molecularweight (measurementerror <10 ppm). Then, on the basis of thesemeasures, a molecular formula iscalculated. Some formulas can correspond to severalcompoundshaving the samemolecularweightbutdifferentchemicalidentity. For high molecularweight, 1 compound can generate up to 3 signals. Batch #1: 115 signalsweredetected, butonly the 29% returned a potentialclassification Batch #2: 173 signalsweredetected, butonly the 43% returned a potentialclassification

31. Results on chemical, proteic and metagenomicanalysis of Infanrixhexa

32. Proteinfractionanalysis According to the manufacturer, InfanrixHexa vaccine contains some proteins. The sample hasbeenanalyzed for the identification of theseproteins. At a visualanalysis, the sample appearsmilky. Differentanalysishavebeenconducted on the sample: 1 st analysis: Digestionasitis To start with, the sample hasbeensubjected to an enzymaticdigestionprocess: 10 μL of a raw sample hasbeentreated with 50 μL Trypsin, left overnight in thermoblockat 37 ° C. A 1 mg / mLhemoglobin control hasbeenprepared and treatedas the sample. Thisanalysisrevealed the absence of anyproteins in the sample. 2 nd Analysis: Digestion of Precipitate The sample hasbeenthensubjected to furtheranalysis by separating, by centrifugation, the liquid part from the solid part of the milkysuspension. All the supernatanthasbeentaken. The remaining precipitate hasbeentreated with 30 μL Trypsin and left overnight in thermoblockat 37 ° C. A 1 mg / mLhemoglobin control hasbeenprepared and treatedas the sample. Afterdigestion, the sample and the control havebeencentrifuged. The supernatanthasbeentaken and placed in vials for analysis. 20 μL osmotized H2O havebeenadded in order to giveenough volume for injection. Thisanalysisrevealed the absence of anyproteins in the sample. 3 rdanalysis: Bradford assay To identify the actualpresence of proteins, InfanrixHexa vaccine hasbeensubjected to the Bradford assay. 200 μL of the raw sample hasbeentreated with 300 μL osmotic H2O to obtain volume. Then 500 μL of Bradford reagenthavebeenadded. After a visualanalysis, we can confirm the presence of proteins or peptide sequencesgiven by the blue color. Based on the calibration line, a proteinconcentration of 1.099 mg/mLwasdetected. 4 thanalysis: Digestionasitisat 57 ° C AfterBradford'sassay, 20 μL of the raw sample hasbeentreated with 80 μL of Trypsin. A 1 mg / mLhemoglobin control hasbeenprepared and treatedas the sample. Theyhavebeenleft in thermoblockat 37 ° C for 4 hours and thenat 57 ° C for 30 minutes. The sample and the control havethenbeensubjected to centrifugation and the supernatanthasbeentaken and placed in vial for analysis. In order to process the data thusobtained, the Mascot database hasbeeninitiallyusedbutnothinghasbeenfound . Therefore, the GMP hasbeenusedbutalso in this case no proteinsequenceshavebeendetected.

33. By the DeNovoresearch, the following peptide sequencesthat do notmeet the trypsincuttingcriteria and thereforepotentiallybelong to free peptideshavebeenidentified. Belowis the detectedsequences list:

34. Metabolicfraction Analysis In Batch # 1 wehave65 signals of which only 35% are known(table 1) Ithasnotbeenpossible to carry out the analyzes on differentbatchesbecausesinceall the InfanrixHexawehavepurchased for more than a year on the nationalterritory, in differentregions and in differentperiods, belong to batch A21CD072D. Moleculespotentiallybelonging to toxincategoryhavebeenresearched. (table 2) Theyhavebeensuggested on the basis of accurate m/z mode research (error <10 ppm) using the toxiccompounds database in the Metlinsearchengine. Furthermore, the presence of formic acid in the form of sodiumsalt and a polymerderiving from contaminations of PolyEthyleneGlycol(PEG) with an averagemolecularweightequal to 1340 Da havebeendetected.

35. Tables of contaminants

36. Table of chemicaltoxines

37. Metagenomicanalysis of Infanrixhexa: Itwasnotpossible to detect the viruses of poliomyelitisbecause the treatment with formaldehyde and glutaraldehyde degradate all the geneticmaterial in the vaccine from VERO cells (continuouscell line potentiallycancerogenic) and the viruses (polio antigens and adventitious agents). EMA answeredthat the immunogenicity is guaranteed by the Antigen D, the proteinexpressed by the polio virusesbeforeinactivation Nextsteps -analysis with Maldi-Tof-MS of Infanrixhexa -fulfill in processanalysis of Hexyon, Gardasil, MMR vax Pro, Vaxelis -quantitative analysis of metals on Infanrixhexa, Hexyon, Priorix tetra and Gardasil