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to the server to view the underlying object.
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"content": "Those who support mandatory vaccination... How far are you prepared for the enforcement to go?!.... How many more vaccines will be added to the schedule? At what point in time do we say no more? What happens when your child gets sick and the Government take YOUR child and forcibly drug them?!.... <br />We are on a slippery slope to forced unlimited drugging with no say in the ingredients, the dose or type... Already some families children have been drugged and/or vaccinated against their will... <br />I don't trust the Government do you? <br />Do you \"think\" our Government REALLY have our best interests at heart?... <br />Before you support \"mandatory\" drugging, segregation and discrimination please think about how this can be used against YOU and your family in the future...<br /><br /><a href=\"https://www.minds.com/search?f=top&t=all&q=vaccineinjuryisreal\" title=\"#vaccineinjuryisreal\" class=\"u-url hashtag\" target=\"_blank\">#vaccineinjuryisreal</a> <a href=\"https://www.minds.com/search?f=top&t=all&q=corruptgovernments\" title=\"#corruptgovernments\" class=\"u-url hashtag\" target=\"_blank\">#corruptgovernments</a> <a href=\"https://www.minds.com/search?f=top&t=all&q=yellowvests\" title=\"#yellowvests\" class=\"u-url hashtag\" target=\"_blank\">#yellowvests</a> <a href=\"https://www.minds.com/search?f=top&t=all&q=vaccinesarenotsafenoreffective\" title=\"#vaccinesarenotsafenoreffective\" class=\"u-url hashtag\" target=\"_blank\">#vaccinesarenotsafenoreffective</a> <a href=\"https://www.minds.com/search?f=top&t=all&q=readavaccineinsert\" title=\"#readavaccineinsert\" class=\"u-url hashtag\" target=\"_blank\">#readavaccineinsert</a> ",
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"published": "2019-01-16T02:05:39+00:00",
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"content": "Those who support mandatory vaccination... How far are you prepared for the enforcement to go?!.... How many more vaccines will be added to the schedule? At what point in time do we say no more? What happens when your child gets sick and the Government take YOUR child and forcibly drug them?!.... \nWe are on a slippery slope to forced unlimited drugging with no say in the ingredients, the dose or type... Already some families children have been drugged and/or vaccinated against their will... \nI don't trust the Government do you? \nDo you \"think\" our Government REALLY have our best interests at heart?... \nBefore you support \"mandatory\" drugging, segregation and discrimination please think about how this can be used against YOU and your family in the future...\n\n#vaccineinjuryisreal #corruptgovernments #yellowvests #vaccinesarenotsafenoreffective #readavaccineinsert ",
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"content": "Excerpt: Read the links to the right and the fifteen PubMed links below. See what you <br />think. If you want more, there are many, many more. You can search <br />PubMed for keywords and read until your eyes are bleary. And it's not just <br />about autism.<br /><br />If you have a child with any autoimmune condition: asthma, allergies, pandas, <br />mitochondria disorder, adhd, diabetes, and so on... sadly, you will find there <br />are links to vaccines for all types of autoimmune disorders.<br /><br />Anecdotal no more. You know where to look. Check it out. Pass it on.<br /><br /><a href=\"http://www.regardingcaroline.com/pubmed.html?fbclid=IwAR1FxjMe_obOQx-hVjjftvijnJAMZJ-XXgRrNO1kVM3EaeXoEZbWhdoBOHs\" target=\"_blank\">http://www.regardingcaroline.com/pubmed.html?fbclid=IwAR1FxjMe_obOQx-hVjjftvijnJAMZJ-XXgRrNO1kVM3EaeXoEZbWhdoBOHs</a> ",
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"published": "2019-01-12T09:36:08+00:00",
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"content": "Excerpt: Read the links to the right and the fifteen PubMed links below. See what you \nthink. If you want more, there are many, many more. You can search \nPubMed for keywords and read until your eyes are bleary. And it's not just \nabout autism.\n\nIf you have a child with any autoimmune condition: asthma, allergies, pandas, \nmitochondria disorder, adhd, diabetes, and so on... sadly, you will find there \nare links to vaccines for all types of autoimmune disorders.\n\nAnecdotal no more. You know where to look. Check it out. Pass it on.\n\nhttp://www.regardingcaroline.com/pubmed.html?fbclid=IwAR1FxjMe_obOQx-hVjjftvijnJAMZJ-XXgRrNO1kVM3EaeXoEZbWhdoBOHs ",
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"content": "The rise of children's hospitals <br /><br />Have you noticed that new children's hospitals are popping up all across the country, and they are expanding every year. Everyone thinking this of course means better treatment but nobody asked the real question...why are so many children sick?<br /><br />Did you know that between 2004 and 2009, hospitals experienced a greater increase in the number of children hospitalized with a chronic condition by 19.2%? The greatest cumulative increase (32.5%) was attributable to children with a significant chronic condition affecting 2 or more body systems. There has been a significant increase in the overall rate of childhood cancers in recent decades, up 27% since 1975. There was an increase in leukemia, which is up almost 35% since 1975. Leukemia is the most common cancer in kids. Soft tissue cancers, like those that develop in bones or muscles, are up nearly 42%. Non-Hodgkin's lymphoma is up 34%.<br /><br />Children are being diagnosed with illnesses such as autism, asthma, allergies, cancer and ADHD at an unbelievable rate. The etiology of autism continues to confound mainstream medicine (deathcare), yet parents, independent researchers, and anyone with a few brains cells dedicated to unraveling the mystery are putting the pieces of the puzzle into place. Mainstream media and medicine are nothing more than prostitutes for the companies and organizations who profit from the sick children. Although the specific pathophysiology of each individual child's illness varies, they all have the same basic underlying causes. It is a perfect storm of poison including decades of pharmaceutical over-usage, toxic food or nutritionally deficient diets, excessive exposure to environmental toxins and without question excessive toxic exposure from vaccination.<br /><br />The only way out of this madness is to opt out of their insane system. Take control of our children's health. Being educated and informed is the only survival mechanism because those in media and medicine are mostly liars who are paid to protect their ruling pharma overlords in order to earn there own self serving compensation. The deception is beyond our wildest imagination and the lies are stacked as far the eyes can see. Reject their toxic food, medications, deathcare and vaccines. Relying on the legal system or political system to enforce change and protect us is just another illusion tricking you into bullshitting yourself that someone else is going to save you, your family or our society. It's up to us...the tipping point has arrived. <br /><br />Do you want healthy children or more children's hospitals? Time to decide where you stand.<br /><br />-Chris Kirkoff ",
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"published": "2019-01-03T23:23:37+00:00",
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"content": "The rise of children's hospitals \n\nHave you noticed that new children's hospitals are popping up all across the country, and they are expanding every year. Everyone thinking this of course means better treatment but nobody asked the real question...why are so many children sick?\n\nDid you know that between 2004 and 2009, hospitals experienced a greater increase in the number of children hospitalized with a chronic condition by 19.2%? The greatest cumulative increase (32.5%) was attributable to children with a significant chronic condition affecting 2 or more body systems. There has been a significant increase in the overall rate of childhood cancers in recent decades, up 27% since 1975. There was an increase in leukemia, which is up almost 35% since 1975. Leukemia is the most common cancer in kids. Soft tissue cancers, like those that develop in bones or muscles, are up nearly 42%. Non-Hodgkin's lymphoma is up 34%.\n\nChildren are being diagnosed with illnesses such as autism, asthma, allergies, cancer and ADHD at an unbelievable rate. The etiology of autism continues to confound mainstream medicine (deathcare), yet parents, independent researchers, and anyone with a few brains cells dedicated to unraveling the mystery are putting the pieces of the puzzle into place. Mainstream media and medicine are nothing more than prostitutes for the companies and organizations who profit from the sick children. Although the specific pathophysiology of each individual child's illness varies, they all have the same basic underlying causes. It is a perfect storm of poison including decades of pharmaceutical over-usage, toxic food or nutritionally deficient diets, excessive exposure to environmental toxins and without question excessive toxic exposure from vaccination.\n\nThe only way out of this madness is to opt out of their insane system. Take control of our children's health. Being educated and informed is the only survival mechanism because those in media and medicine are mostly liars who are paid to protect their ruling pharma overlords in order to earn there own self serving compensation. The deception is beyond our wildest imagination and the lies are stacked as far the eyes can see. Reject their toxic food, medications, deathcare and vaccines. Relying on the legal system or political system to enforce change and protect us is just another illusion tricking you into bullshitting yourself that someone else is going to save you, your family or our society. It's up to us...the tipping point has arrived. \n\nDo you want healthy children or more children's hospitals? Time to decide where you stand.\n\n-Chris Kirkoff ",
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"content": "<a href=\"https://www.reddit.com/r/conspiracy/comments/1cm0t3/original_research_the_mountain_of_evidence_for_a/\" target=\"_blank\">https://www.reddit.com/r/conspiracy/comments/1cm0t3/original_research_the_mountain_of_evidence_for_a/</a> ",
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"published": "2019-01-03T23:23:02+00:00",
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"content": "https://www.reddit.com/r/conspiracy/comments/1cm0t3/original_research_the_mountain_of_evidence_for_a/ ",
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"content": "<br />Our government is SO desperate to poison and brainwash us, and SO terrified of us woke individuals waking up the rest of you, that they’ve decided to call sanity insanity. According to the government, it’s now insanity to want to be healthy and not allow our government to drug and poison us. <br />Don’t drink the fluoride, folks. <br /><br />From Waking Times: \"\"Now, you suddenly may have a mental disorder, at least according to scientists at the University of Northern Colorado who conducted a case study about the obsession of eating healthy. This new eating disorder is called orthorexia nervosa (ON) and is said to be driven by a fear of being unhealthy and disgust for low-quality food.<br /><br />“Orthorexia nervosa (ON) is a term introduced to describe a condition characterized by a pathologic obsession for bio-logically pure and healthy nutrition.” ~Ryan M. Moroze, MD. et al [1]<br />The psychologists conducting the study argue that healthy eating can become dangerous if one becomes fixated on the types of ingredients in food, how the food is cooked, and what materials are used to prepare it. Those “suffering” from orthorexia may take extra time to prepare their food and carefully consider what they are willing to eat.\" <a href=\"https://www.minds.com/search?f=top&t=all&q=BigPharma\" title=\"#BigPharma\" class=\"u-url hashtag\" target=\"_blank\">#BigPharma</a>",
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"published": "2019-01-03T14:11:10+00:00",
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"content": "\nOur government is SO desperate to poison and brainwash us, and SO terrified of us woke individuals waking up the rest of you, that they’ve decided to call sanity insanity. According to the government, it’s now insanity to want to be healthy and not allow our government to drug and poison us. \nDon’t drink the fluoride, folks. \n\nFrom Waking Times: \"\"Now, you suddenly may have a mental disorder, at least according to scientists at the University of Northern Colorado who conducted a case study about the obsession of eating healthy. This new eating disorder is called orthorexia nervosa (ON) and is said to be driven by a fear of being unhealthy and disgust for low-quality food.\n\n“Orthorexia nervosa (ON) is a term introduced to describe a condition characterized by a pathologic obsession for bio-logically pure and healthy nutrition.” ~Ryan M. Moroze, MD. et al [1]\nThe psychologists conducting the study argue that healthy eating can become dangerous if one becomes fixated on the types of ingredients in food, how the food is cooked, and what materials are used to prepare it. Those “suffering” from orthorexia may take extra time to prepare their food and carefully consider what they are willing to eat.\" #BigPharma",
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"content": "<a href=\"https://gumshoenews.com/2018/12/07/world-first-george-h-w-bush-executed-by-military-tribunal/?fbclid=IwAR230-iV-PrUi3gBF8U4Cg5dA5QqTogbxB5Ya5wkZpIIBsCsifUGRO2_T7A\" target=\"_blank\">https://gumshoenews.com/2018/12/07/world-first-george-h-w-bush-executed-by-military-tribunal/?fbclid=IwAR230-iV-PrUi3gBF8U4Cg5dA5QqTogbxB5Ya5wkZpIIBsCsifUGRO2_T7A</a> ",
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"url": "https://www.minds.com/newsfeed/924988822140993536",
"published": "2018-12-27T11:35:20+00:00",
"source": {
"content": "https://gumshoenews.com/2018/12/07/world-first-george-h-w-bush-executed-by-military-tribunal/?fbclid=IwAR230-iV-PrUi3gBF8U4Cg5dA5QqTogbxB5Ya5wkZpIIBsCsifUGRO2_T7A ",
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"content": "<a href=\"https://www.jeffereyjaxen.com/blog/the-tale-of-the-worthless-vaccine?fbclid=IwAR1kzdgfzvNjewg63ZmE2HV4r4wMRYMNHlgJM7cyiW_d9TSuKaEt03n3PLw\" target=\"_blank\">https://www.jeffereyjaxen.com/blog/the-tale-of-the-worthless-vaccine?fbclid=IwAR1kzdgfzvNjewg63ZmE2HV4r4wMRYMNHlgJM7cyiW_d9TSuKaEt03n3PLw</a> ",
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"published": "2018-12-25T08:42:36+00:00",
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"content": "https://www.jeffereyjaxen.com/blog/the-tale-of-the-worthless-vaccine?fbclid=IwAR1kzdgfzvNjewg63ZmE2HV4r4wMRYMNHlgJM7cyiW_d9TSuKaEt03n3PLw ",
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{
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"content": "Do you really know what is in the MMRV vaccine?<br />Of course we don’t, well now we do and you should be furious! Read the last paragraph first... <br /><br />Vaccinegate:<br />Metagenomic analysis report on Priorix Tetra<br /><br />Introduction<br /><br />As is known, vaccines are biological drugs used for the prevention of some infectious diseases and are made up of several components: antigens (viruses, inactivated or attenuated bacteria, inactivated toxins, proteins or complex molecules derived from viruses and bacteria, able to stimulate the immune response), adjuvants (substances that increase the capacity of the vaccine antigens to induce the antibody immune response), the excipients (substances necessary to formulate the vaccine, or to preserve it from bacterial contamination) and contaminations (trace substances from raw materials, eg cell lines for bacteria and virus growth, or from the processing process, eg formaldehyde, antibiotics). During the registration phase of a biological drug, the vaccine is subjected to the controls provided by the EMA guidelines and agreed with the regulatory body according to the specific type of vaccine. These checks are then carried out on a representative number of samples on each lot before marketing.<br />The responsibility for the conformity of the product sold is therefore of the producer and of the regulatory bodies in charge of the control.<br />Since the safety of a vaccine depends on its compliance with the quality criteria, especially regarding the control of the absence of toxic or potentially toxic contamination (ie for which no human effects are known) it is of great importance that such compliance is respected in a very strict manner.<br /><br />Various studies in the literature have posed the problem of the presence of various types of contaminations, both chemical and microbiological, thus opening the question if the vaccines actually comply with the directives imposed by the regulatory bodies, if in turn the regulatory bodies apply the control for the respect of these directives and if the regulatory bodies have defined with effective guidelines the criteria for the control and containment of such contaminations. To answer these questions, Corvelva commissioned the analysis of biological contaminations, which should never be present in vaccines, at a highly qualified center of services specializing in the genomic sequencing of DNA and RNA.<br /><br />The study commissioned by Corvelva was based on two types of analysis:<br /><br />1. Test of presence of nucleic acids (DNA / RNA) of human and animal origin and of microorganisms (viruses, bacteria) using the Next Generation Sequencing method, which allowed to quantify in a highly specific and accurate manner the sequence of genetic material contained in the vaccines examined<br /><br />2. Verification of the correspondence of the genome sequences of live attenuated or inactivated bacteria and viruses present in the vaccines (presence of genetic variants)<br />Description of the method used for the analysis<br />Next Generation Sequencing, also known as deep sequencing, generates a single sequence from each DNA fragment, or cDNA, present in a sample. The downstream bioinformatics analysis then allows the differentiation between the origin of the sequence fragments, for example human, bacterial species or a particular virus. This means that mixed biological samples can be easily solved with this technology, which has now entered the routine of genomic research and diagnostics. Moreover, from NGS data it is possible to reconstruct the entire sequence of viral DNA and RNA genomes and bacterial genomes present in the sample and compare it with the reference genomes present in public databases.<br /><br />The samples examined are shown below along with the results obtained, grouping them by classes of similar vaccines:<br /><br />* ssRNA: single strand RNA, single-stranded RNA; dsDNA: double strand DNA, double stranded DNA. The underlined terms are made up or contain genetic material (DNA and / or RNA)<br /><br />Lot # 1 - A71CB205A and Lot # 2 - A71CB256A<br />Product Name: Product Type: Manufacturer: Composition: 1<br />Priorix Tetra<br />Lots analyzed<br />Tetravalent measles, mumps, rubella, varicella vaccine<br />GlaxoSmithKline, Belgium<br />live attenuated viruses: Measles (ssRNA) Swartz strain, cultured in embryo chicken cell cultures; Mumps (ssRNA) strain RIT 4385, derived from the Jeryl Linn strain, cultured in embryo chicken cell cultures; Rubella (ssRNA) Wistar RA 27/3 strain, grown in human diploid cells (MRC-5); Varicella (dsDNA) OKA strain grown in human diploid cells (MRC-5)<br /><br />Required analysis<br />Test of presence of nucleic acids (DNA / RNA) of human and animal origin and of microorganisms (viruses, bacteria), using a metagenomic / metatrascritomic type approach on the Illumina platform of Next Generation Sequencing.<br />From the comparison of these three vaccines it is possible to highlight the following criticalities:<br />The Priorix Tetra is the vaccine with the highest amount of extraneous DNA contaminant (total DNA extracted from 3.7 μg to 1.7 μg, of which 88% is human, then from the MRC-5 cells, and the remaining 12% comes from adventitious microorganisms , such as viruses, bacteria, worms).<br /><br />Human genomic DNA is high molecular weight above 60,000 bp. and the total sequential coverage of the entire reference human genome (HG-19) shows that it is the entire genome of the fetal cells used for the cultivation of vaccine viruses to be present and not only portions thereof.<br /><br />From the EMA's answer to our question2 on the limits imposed on the residues of foreign genetic material in vaccines, it appears that in fact there are no limits for each vaccine but only for some, reported in the monographs of the product; The maximum limit envisaged ranges from 10 pg to 10 ng, based on the theoretical calculation of the possibility of foreign genomic DNA to cause oncogenic mutations.<br /><br />It is noteworthy that regulatory authorities do not require that these contaminations are tested in the final product, but only in the initial preparation phase, and that for the attenuated virus vaccines the purification of these contaminations is a critical step3. The EMA has not provided specific studies on the dangers of fetal residual DNA, which allow to assess the risk to human health of these contaminations, so this limit remains arbitrary today.<br /><br />It follows that for these two lots of Priorix Tetra is about 140 times higher than the maximum limit of 10 ng and 140,000 times higher than the minimum limit of 10 pg.<br /><br />On the question of contaminating human DNA, the World Health Institute in an official 2011 document entitled 'Recommendations for the Evaluation of Animal Cells and Subsidies for the Production of Biological Products and Cells' argues that what is necessary to take into consideration with respect to rcDNA (residual cell DNA) in vaccines is:<br /><br />A. a reduction in the amount of contaminating DNA during the manufacturing process;<br /><br />B. a reduction in the size of the contaminating DNA during the manufacturing process;<br /><br />C. a chemical inactivation of the biological activity of DNA occurred during the manufacturing process.<br /><br />Taking into account the three requests described above, the product is considered by their regulatory organs (NRAs) and control laboratories (NLC), to be at an acceptable level of risk regarding the presence of DNA from the cell substrate, based on (a) and / or (b) and / or (c), when the data show that adequate levels of safety have been achieved.<br />In particular in the 2 batches of Priorix Tetra vaccine tested to date, point A. it does not occur because the quantity is about 140 times higher than that recommended by the FDA (in Briefing Document September 19, 2012: Vaccines and Related Biological Products Advisory Committee Meeting) and by the EMA, ie ≤ 10ng per dose; point b) does not occur because the DNA is high molecular weight (most> 10,000 bp, as can easily be verified using a simple agarose gel to control the quality of the DNA extracted from the vaccine), ie 50 times greater than the size recommended by the FDA (200bp or less). Finally, in the same vaccine, point c) does not occur because, containing attenuated viruses, a possible chemical inactivation of DNA, it would also inactivate viruses.<br /><br />Priorix Tetra lot comparison<br />Lot # 1 - A71CB205A Lot # 2 - A71CB256A DNA analysis<br />Total DNA extracted: 1.7 μg total per 0.5mL dose<br />Performed DNA-seq analysis using a metagenomic approach, suuntotaledi3 .830.074 of sequences produced<br /><br />Present genomic DNA of:<br />Varicella1 4%<br />Pollo 4% <br />Man (MRC-5) 74% <br />proteobacteria 1% <br />RNA virus 0.01% not assigned <br />5%DNA analysis<br /><br />Total DNA extracted: 3.7 μg total per 0.5mL dose<br />Performed DNA-seq analysis using a metagenomic approach, suuntotaledi5 .836.297 of sequences produced.<br /><br />Present genomic DNA of:<br />Varicella1 4% <br />Man (MRC-5) 8 8% (about 3.3 μg equivalent to about 300,000 fetal cells) 0.0003% <br />RNA virus not assigned <br />0.5%RNA analysis<br /><br />Total RNA extracted:<br />not quantifiable with standard fluorimetric methods<br /><br />Performed RNA-seq analysis using a metatrascritomic approach,<br />on a total of 10.445.038 of sequences produced.<br />Measure<br />0.004% Mumps <br />0.008% Rubella<br />0.00007% Varicella<br />5% other viruses <br />about 0.002% helminth <br />0.6% P o l l o <br />0. 2% for men <br />87%<br />not allocated 5%<br /><br />RNA analysis<br /><br />Total RNA extracted: 200ng per 0.5mL dose<br />Performed RNA-seq analysis using a metatrascritomic approach,<br />out of a total of 6,171,266 of produced sequences.<br />Measles 0.004% <br />Mumps 0.008% <br />Rosolian nd% * <br />Varicella 7%<br />other viruses about 0.001%<br />nematoda 1.50% <br />Proteobacteria 5.5%<br />U or m o 6 8%<br />not allocated 6%<br />* sequencing with 260.343.42 reads: 114 reads equal to 0.00004%<br /><br />Methods and results<br /><br />DNA and RNA extraction Priorix Tetra lot. A71CB205A<br />The lotA71CB205A lot was tried in June 2018.<br />Genomic DNA extraction was carried out with the Maxwell® 16 Blood DNA Purification Kit sold by Promega and with the automatic extractor Maxwell® 16 IVD (Promega), following the manufacturer's protocol. RNA extraction was performed using the PureLinkTM Viral RNA / DNA Kit Mini Kit (Invitrogen) following the manufacturer's protocol.<br /><br />The starting quantities used for the extractions are as follows (starting from a single vial of product):<br />● DNA extraction: 125 μl on 500 μl total of suspension for injection<br />● RNA extraction: 125 μl on 500 μl total of suspension for injection<br /><br />The quantification and quality control of the extracted DNA and RNA was carried out with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).<br />Below is the result of the quantifications (ND = NanoDrop 1000; QB = Qubit 2.0; HS = dsDNA HS Assay Kit)<br /><br />The measurement of DNA concentration with QuBit fluorometer showed that the lot A71CB205A contains a quantity of gDNA of 1.7 μg total per dose of 0.5mL, calculated as follows:<br />9.41ng / μl (concentration determined at QuBit) x 45 (final resuspension volume of DNA after extraction, expressed in microliters) x 4 (the starting volume subjected to the extraction procedure is 1/4 of the volume of the dose contained in the whole vial equal to 0.5mL).<br /><br />DNA and RNA extraction Priorix Tetra lot. A71CB256A<br />Lot A71CB206A was tried in December 2018. Some improvements were made in the procedure, such as:<br />1. Use of a higher volume of injectable starting solution in order to increase the amount of extracted RNA (in the extraction from the previous batch the amount of RNA obtained was below the detection threshold with fluorimeter);<br />2. Performed an electrophoresis in the pulsed field, to get a greater detail on the size of the whole genomic DNA present in the sample;<br />3. Used a measurement mode for the most sensitive RNA (Agilent RNA 6000 Pico Kit on Agilent Bioanalyzer).<br /><br />Genomic DNA extraction was carried out with the Maxwell® 16 Blood DNA Purification Kit sold by Promega and with the automatic extractor Maxwell® 16 IVD (Promega), following the manufacturer's protocol. RNA extraction was performed using the PureLinkTM Viral RNA / DNA Kit Mini Kit (Invitrogen) following the manufacturer's protocol.<br /><br />The starting quantities used for the extractions are as follows, starting from two vials of the product of the same lot:<br />● DNA extraction: 300μl on 500μl suspension for injection (ampoule 1)<br />● RNA extraction: the entire volume of powder in a vial was resuspended in 200 μl of physiological saline supplied in the package, instead of 500 μl, and the entire volume was used for<br />RNA extraction (vial 2).<br /><br />The quantification and quality control of the extracted DNA was carried out respectively with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and with the spectrophotometer N anoDrop1000 (ThermoFisherScientific, Waltham, USA).<br />Following is the result of DNA quantifications (ND = NanoDrop 1000; QB = Qubit 2.0; HS = dsDNA HS Assay Kit)<br /><br />The measurement of DNA concentration with the QuBit fluorometer showed that the lot A71CB256A contains an amount of gDNA of 3.7 μg total per dose of 0.5mL, calculated as follows:<br />40.8 ng / μl (concentration determined at QuBit) x 55 (final resuspension volume of DNA after extraction expressed in microliters) x 5/3 (the initial volume subjected to the extraction procedure is 300 μl on 500 μl of suspension) .<br />Electrophoresis in pulsed field (PFGE, 5-80Kb, stroke 14h in TBE 0.5x, 80V) of DNA extracted from lot A71CB256A, made visible thanks to SybrGreen fluorescent interlayer, highlighted the presence of a wide 'strip' of DNA genomic that reaches up to very high molecular weights, but with a significant amount of DNA in the 20-60Kbp range. In particular in the photo below, the sample ID1205_PT _raw is the lysate of the material contained in the vaccine before DNA purification, while PT_gDNA is the genomic DNA after extraction; Mid, Low and Lambda are 3 commercial molecular weight markers.<br />The quantification and quality control of the extracted RNA was performed on Agilent 2100 Bioanalyzer using the A gilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA). Below are the concentration values, the RNA (Integrity Number) measured at Bionalyzer:<br /><br />The amount of RNA contained in the lot A71CB256A vaccine vial was found to be about 200ng.<br />The RIN of 8 indicates an RNA of excellent quality and an intact eukaryotic RNA, being present both the 18S and 28S peaks typical of eukaryotic RNA:<br />Preparation of the DNA-seq library with Illumina technology<br />The Ovation® Ultralow System V4 1-96 kit (Nugen, San Carlos, CA) was used to prepare the libraries, following the manufacturer's indications, starting from 10ng of genomic DNA. The final libraries were quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and tested for quality with the Caliper GX system (PerkinElmer, Waltham, MA) for the lot A71CB205A and Agilent 2100 Bioanalyzer, DNA High Sensitivity Analysis kit (Agilent technologies, Santa Clara , CA) for the lot A71CB256A. <br /><br />Following are the traces for the two libraries obtained:<br />Preparation of the RNA-seq library with Illumina technology<br />For the preparation of the RNA-seq libraries the Ovation® RNA-Seq System V2 kit (Nugen, San Carlos, CA) was used to prepare the cDNA and the Ovation® Ultralow System V4 1-96 kit to prepare the library starting from 10ng of cDNA. The final libraries were quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and tested for quality with the Caliper GX system (PerkinElmer, Waltham, MA) for the lot A71CB205A and Agilent 2100 Bioanalyzer, DNA High Sensitivity Analysis kit (Agilent technologies, Santa Clara, CA) for the lot A71CB256A. <br /><br />Following are the traces for the two libraries obtained:<br /><br />In order to validate the workflow from the preparation of the library to the analysis of the data, an ATCC standard (mix of genomic DNA with a known composition, 20 Strain Staggered Mix Genomic Material, ATCC® MSA-1003TM) was used to construct a library with the kit Ovation®UltralowSystemV4 1-96 starting from 10ng of DNA. Following is the path to the Bioanalyzer of the library obtained:<br /><br />Sequencing<br /><br />The libraries have been sequenced on Illumina HiSeq2500 instrument in 125bp 'paired-end' mode, according to Illumina standard indications. Version 1.8.2 of the Illumina CASAVA pipeline was used to process raw sequences.<br /><br />Bioinformatic analysis<br /><br />Cleaning of sequences (trimming)<br />The sequences of the adapters (ie 'artificial' oligonucleotide sequences that are introduced during the Illumina library preparation) and the low quality read DNA bases have been removed using the ERNE1 and Cutadapt2 software.<br />Identification of organisms of origin of DNA and cDNA sequences Metagenomic analysis was performed using the Kraken3 software on the 'Human-Virus-Bacteria_25mer' database<br />(h ttps: //ccb.jhu.edu/software/kraken/).<br /><br />Kraken is a classifier that assigns taxonomic labels to short DNA readings. It does this by examining the k-mers in a reading and querying a database with those k-mers.<br /><br />Bibliographic references<br />1. Del Fabbro, C et al. 2013 An evaluation of readings on Illumina NGS data analysis. Del Smith C, Scalabrin S, Morgante M, Giorgi FM. PLoS One. 2013 Dec 23; 8 (12): e85024. doi: 10.1371 / journal.pone.0085024. eCollection 2013<br />2. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, [S.l.], 17 (1): 10-12 (2011). ISSN 2226-6089. Date accessed: 02 Apr. 2015. doi: http: //dx.doi.org/10.14806/ej.17.1.200 paper<br />3. Wood and Salzberg. Kraken: ultrafastmetagenomicsequence classification using exact alignments Genome Biology 2014, 15: R46<br /><br />From the comparison of these two vaccines it is possible to highlight the following critical issues:<br />The Priorix Tetra is a vaccine with a high amount of contaminating foreign DNA of which on average 80% is human, therefore coming from MRC-5 cells; in Lot # 1 there is also 4% of DNA coming from embryo chicken cells; the remaining 20% belongs to viruses (retroviruses, infectious viruses, and carcinogens, phages) and adventitious microorganisms such as bacteria, and worms; in the Priorix Tetra vaccine the human genomic DNA is high molecular weight (> 10.000bp) and the total sequential coverage of the entire reference human genome (HG-19) shows that it is the entire genome of the fetal cells used for the culture of vaccine viruses to be present and not just portions of it.<br /><br />From the EMA's response to our question6 on the limits imposed on residues of foreign genetic material in vaccines, it appears that in fact there are no limits for each vaccine but only for some, reported in the monographs of the product; The maximum limit envisaged ranges from 10 pg 7 to 10 ng, based on the theoretical calculation of the possibility of foreign genomic DNA to cause oncogenic mutations.<br /><br />It is noteworthy that regulatory authorities do not require that these contaminations be tested in the final product, but only in the initial preparation phase, and that for the attenuated virus vaccines the purification of these contaminations is a critical step8. The EMA has not provided specific studies on the dangers of fetal residual DNA, which allow to assess the risk to human health of these contaminations, so this limit remains arbitrary today.<br /><br />In Lot # 2 of Priorix Tetra, fetal DNA is about 325 times higher than the maximum limit of 10 ng and 325,000 times higher than the minimum limit of 10 pg.<br /><br />On the question of contaminating human DNA, the World Health Institute in an official 2011 document entitled 'Recommendations for the Evaluation of Animal Cells and Subsidies for the Production of Biological Products and Cells' argues that what is necessary to take into consideration with respect to rcDNA (residual cell DNA) in vaccines is:<br /><br />A. a reduction in the amount of contaminating DNA during the manufacturing process;<br /><br />B. a reduction in the size of the contaminating DNA during the manufacturing process;<br /><br />C. a chemical inactivation of the biological activity of DNA occurred during the manufacturing process.<br /><br />Taking into account the three requests described above, the product is considered by their regulatory organs (NRAs) and control laboratories (NLC), to be at an acceptable level of risk regarding the presence of DNA from the cell substrate, based on (a) and / or (b) and / or (c), when the data show that adequate levels of safety have been achieved.<br /><br />In particular in the 2 batches of Priorix Tetra vaccine tested to date, point A. it does not occur because the quantity is about 140 times higher than that recommended by the FDA (in Briefing Document September 19, 2012: Vaccines and Related Biological Products Advisory Committee Meeting) and the EMA, ie ≤ 10ng per dose; point b) does not occur because the DNA is high molecular weight (most> 10,000 bp, as can easily be verified using a simple agarose gel to control the quality of the DNA extracted from the vaccine), ie 50 times greater than the size recommended by the FDA (200bp or less). Finally, in the same vaccine, point c) does not occur because, containing attenuated viruses, a possible chemical inactivation of DNA, it would also inactivate viruses.<br /><br />Analysis of Genetic Variants<br /><br />With Next Generation Sequencing technology it is possible to reconstruct the entire sequence of viral DNA and RNA genomes and bacterial genomes present in the sample and compare it with the reference genomes present in public databases. The technology can therefore also allow to monitor in time how and if the sequence of a viral or bacterial genome changes during the production procedure of a vaccine.<br /><br />The result of the variants call (single nucleotide and small insertions / deletions) compared to the reference strains found in public databases (NCBI, National Center for Biotechnology Information, <a href=\"https://www.ncbi.nlm.nih.gov/\" target=\"_blank\">https://www.ncbi.nlm.nih.gov/</a>) carried out in the vaccine samples containing live attenuated virus or bacteria gave the following results:<br /><br />Sample 1. - Priorix Tetra<br /><br />1. The genome of the measles virus contained in the vaccine is identical to the sequence of the Edmonston Swartz strain deposited in the databases having accession number AF266291.1.<br />The number of variants detected was in fact equal to 0;<br /><br />2. The genome of the mumps virus contained in the vaccine showed a single mutation compared to the Jeryl-Lynn viral strain present in public databases with the accession number AF338106.1;<br /><br />3. The genome of the rubella virus has not been detected;<br /><br />4. The genome of the varicella virus contained in the vaccine has shown four mutations compared to the Human herpesvirus 3<br />present in public databases with access number AB097932.1;<br /><br />The sequence of viral antigens / genomes is a strictly confidential data that is not provided by the EMA. There are no guidelines that regulate the analysis of genetic mutations and the study of effects on human health.<br /><br />The high frequency of genetic mutations in viruses and bacteria, as well as in the DNA of cell lines in culture, is a problem of great importance with regard to safety, as it is not known how any variants found can modify the infectious capacity and the stimulation of the immune system towards autoimmune reactions.<br /><br />For example, it is suggested that Efsa now requires the genomic characterization of probiotic strains for human / animal use and subsequently the demonstration of the coincidence, over time, of the sequence of the microorganism compared to that declared, while in the case of vaccines, as Vivotif, are tolerated as many as 154 genetic variants compared to that stated in the data sheet and present in public databases as a reference vaccine strain.<br /><br />The presence of genetic variants in vaccine samples compared to the strains declared can be considered in our opinion a non-compliance of drugs.<br /><br />Deepening<br /><br />In the recent prepublished article on F1000research \"Do you cov me? NGS technology has been used to analyze biological matrices of different types, including two batches of measles-mumps-rubella-varicella vaccine. PRIORIX TETRA (GlaxoSmithKline SpA), with the aim of demonstrating how, even from low-coverage NGS sequencing (ie of a few hundred thousand sequence fragments to 1 million), it is possible to characterize the biological component in a complex matrix. The next-generation sequencing was already used on vaccine samples in the publication \"Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis\" to demonstrate the coincidence between the genome of the vaccine virus of mumps and the virus found in the brain tissue of an 18-month-old child with SCID who died of encephalitis.<br /><br />In particular, in the pre-published article on F1000research, it is observed that about 80% of the sequences obtained with NGS technology on the two vaccine samples, consisting of human DNA, as impurity present in the working process; the total amount of DNA extraneous is about 2 micrograms, coming from the human fetal cell line MRC-5 used to grow rubella and varicella viruses). The metagenomic analysis performed on these two samples highlights the potential presence of human DNA in all vaccines containing viruses grown in human fetal lines, also already verified with technology other than NGS also by Dr. Deisher in \"Epidemiologic and Molecular Relationship Between Vaccine Manufacture and Autism Spectrum Disorder Prevalence \".<br /><br />Dr. Theresa Deisher's group in the article \"Insertional mutagenesis and autoimmunity induced disease caused by human fetal and retroviral residual toxins in vaccines\" states that the levels of residual DNA in MPR, chickenpox and hepatitis A vaccines available in the United States are abundant the limit set in the current WHO DNA Guideline from immortalized cell lines of 10 ng per dose of vaccine. Although the EMA guideline does not provide for maximum limits for the fetal DNA remaining in the vaccines, Prof. Deisher's group has also taken as reference the maximum dose of 10 ng as a consequence of the fact that the short fragments of fetal DNA present in the vaccines present the ability to integrate into the host DNA and can lead to mutagenesis and / or genomic instability as well as an autoimmune response. Furthermore, in some varicella and MPR vaccines, the presence of fragments of endogenous human retrovirus K (HERVK) has been found, which can be re-activated and can facilitate the integration of free DNA into the host genome.<br /><br />As stated by Dr. Deisher, the danger of retroviral fragments and residual human diploid DNA has not yet been studied in the recipients of the vaccine, although the scientific literature clearly demonstrates the high probability of the dangers of autoimmune and / or insertional mutagenesis due to presence of these residues, and this is a risk to human health that undoubtedly requires serious scientific and epidemiological research.<br />These results are integrated with the new data on the analysis of a new sample of Priorix tetra and the reanalysis of the previous ones, as a non-conformity has emerged concerning the vaccine antigens, ie the dubious presence of the rubella antigen. From the in-depth analysis of the three lots it was confirmed that the rubella antigen is not present, because the number of copies per sample is totally negligible to have an immunostimulatory effect.<br /><br />This more in-depth investigation has made it possible to detect the presence of adventitious DNA and RNA, ie from viruses, bacteria, fungi and helminths in quantities below the limit of detection of the instrument, and therefore in residual quantities. However, it should be emphasized that the average total amount of total DNA varies from 1.9 to 3.7 micrograms, of which about 80% comes from human fetal DNA and the remaining 20% from embryo chicken DNA and adventitious genetic material, then in quantity cumulative non-residual.<br /> <br />It should be noted that in the various lots a considerable variability of the adventitious contaminants was noted, but overall the following categories of adventitious microorganisms were found:<br /><br />■ Bacteria <br />■ Proteobacteria<br />■ Worms <br />■ Platyhelminthes <br />■ Nematoda<br />■ dsDNA viruses <br />■ Varicella virus<br />■ ssRNA viruses <br />■ Rubella, mumps and measles virus<br />■ Other viruses including Influenza A, Cupixi mammarenavirus, Pneumoviridae, Jamestown Canyon, Hepacivirus C, Kobuvirus Enterovirus, Porcine reproductive and respiratory syndrome virus<br />■ Retrovirus<br />■ human and avian endogenous retroviruses<br />■ avian viruses<br />■ human and monkey immunodeficiency viruses <br />■ murine viruses<br />■ Infectious horse anemia virus <br />■ Lymphoproliferative disease virus <br />■ Rous sarcoma virus<br />■ Other viruses <br />■ Human alphaherpesvirus 3 <br />■ Hepatitis b virus<br />■ Yeast virus<br /><br /><a href=\"https://drive.google.com/file/d/1mufQ9Ueoph4T4BufJ71M6v95KvOfhXAw/view\" target=\"_blank\">https://drive.google.com/file/d/1mufQ9Ueoph4T4BufJ71M6v95KvOfhXAw/view</a> ",
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"content": "Do you really know what is in the MMRV vaccine?\nOf course we don’t, well now we do and you should be furious! Read the last paragraph first... \n\nVaccinegate:\nMetagenomic analysis report on Priorix Tetra\n\nIntroduction\n\nAs is known, vaccines are biological drugs used for the prevention of some infectious diseases and are made up of several components: antigens (viruses, inactivated or attenuated bacteria, inactivated toxins, proteins or complex molecules derived from viruses and bacteria, able to stimulate the immune response), adjuvants (substances that increase the capacity of the vaccine antigens to induce the antibody immune response), the excipients (substances necessary to formulate the vaccine, or to preserve it from bacterial contamination) and contaminations (trace substances from raw materials, eg cell lines for bacteria and virus growth, or from the processing process, eg formaldehyde, antibiotics). During the registration phase of a biological drug, the vaccine is subjected to the controls provided by the EMA guidelines and agreed with the regulatory body according to the specific type of vaccine. These checks are then carried out on a representative number of samples on each lot before marketing.\nThe responsibility for the conformity of the product sold is therefore of the producer and of the regulatory bodies in charge of the control.\nSince the safety of a vaccine depends on its compliance with the quality criteria, especially regarding the control of the absence of toxic or potentially toxic contamination (ie for which no human effects are known) it is of great importance that such compliance is respected in a very strict manner.\n\nVarious studies in the literature have posed the problem of the presence of various types of contaminations, both chemical and microbiological, thus opening the question if the vaccines actually comply with the directives imposed by the regulatory bodies, if in turn the regulatory bodies apply the control for the respect of these directives and if the regulatory bodies have defined with effective guidelines the criteria for the control and containment of such contaminations. To answer these questions, Corvelva commissioned the analysis of biological contaminations, which should never be present in vaccines, at a highly qualified center of services specializing in the genomic sequencing of DNA and RNA.\n\nThe study commissioned by Corvelva was based on two types of analysis:\n\n1. Test of presence of nucleic acids (DNA / RNA) of human and animal origin and of microorganisms (viruses, bacteria) using the Next Generation Sequencing method, which allowed to quantify in a highly specific and accurate manner the sequence of genetic material contained in the vaccines examined\n\n2. Verification of the correspondence of the genome sequences of live attenuated or inactivated bacteria and viruses present in the vaccines (presence of genetic variants)\nDescription of the method used for the analysis\nNext Generation Sequencing, also known as deep sequencing, generates a single sequence from each DNA fragment, or cDNA, present in a sample. The downstream bioinformatics analysis then allows the differentiation between the origin of the sequence fragments, for example human, bacterial species or a particular virus. This means that mixed biological samples can be easily solved with this technology, which has now entered the routine of genomic research and diagnostics. Moreover, from NGS data it is possible to reconstruct the entire sequence of viral DNA and RNA genomes and bacterial genomes present in the sample and compare it with the reference genomes present in public databases.\n\nThe samples examined are shown below along with the results obtained, grouping them by classes of similar vaccines:\n\n* ssRNA: single strand RNA, single-stranded RNA; dsDNA: double strand DNA, double stranded DNA. The underlined terms are made up or contain genetic material (DNA and / or RNA)\n\nLot # 1 - A71CB205A and Lot # 2 - A71CB256A\nProduct Name: Product Type: Manufacturer: Composition: 1\nPriorix Tetra\nLots analyzed\nTetravalent measles, mumps, rubella, varicella vaccine\nGlaxoSmithKline, Belgium\nlive attenuated viruses: Measles (ssRNA) Swartz strain, cultured in embryo chicken cell cultures; Mumps (ssRNA) strain RIT 4385, derived from the Jeryl Linn strain, cultured in embryo chicken cell cultures; Rubella (ssRNA) Wistar RA 27/3 strain, grown in human diploid cells (MRC-5); Varicella (dsDNA) OKA strain grown in human diploid cells (MRC-5)\n\nRequired analysis\nTest of presence of nucleic acids (DNA / RNA) of human and animal origin and of microorganisms (viruses, bacteria), using a metagenomic / metatrascritomic type approach on the Illumina platform of Next Generation Sequencing.\nFrom the comparison of these three vaccines it is possible to highlight the following criticalities:\nThe Priorix Tetra is the vaccine with the highest amount of extraneous DNA contaminant (total DNA extracted from 3.7 μg to 1.7 μg, of which 88% is human, then from the MRC-5 cells, and the remaining 12% comes from adventitious microorganisms , such as viruses, bacteria, worms).\n\nHuman genomic DNA is high molecular weight above 60,000 bp. and the total sequential coverage of the entire reference human genome (HG-19) shows that it is the entire genome of the fetal cells used for the cultivation of vaccine viruses to be present and not only portions thereof.\n\nFrom the EMA's answer to our question2 on the limits imposed on the residues of foreign genetic material in vaccines, it appears that in fact there are no limits for each vaccine but only for some, reported in the monographs of the product; The maximum limit envisaged ranges from 10 pg to 10 ng, based on the theoretical calculation of the possibility of foreign genomic DNA to cause oncogenic mutations.\n\nIt is noteworthy that regulatory authorities do not require that these contaminations are tested in the final product, but only in the initial preparation phase, and that for the attenuated virus vaccines the purification of these contaminations is a critical step3. The EMA has not provided specific studies on the dangers of fetal residual DNA, which allow to assess the risk to human health of these contaminations, so this limit remains arbitrary today.\n\nIt follows that for these two lots of Priorix Tetra is about 140 times higher than the maximum limit of 10 ng and 140,000 times higher than the minimum limit of 10 pg.\n\nOn the question of contaminating human DNA, the World Health Institute in an official 2011 document entitled 'Recommendations for the Evaluation of Animal Cells and Subsidies for the Production of Biological Products and Cells' argues that what is necessary to take into consideration with respect to rcDNA (residual cell DNA) in vaccines is:\n\nA. a reduction in the amount of contaminating DNA during the manufacturing process;\n\nB. a reduction in the size of the contaminating DNA during the manufacturing process;\n\nC. a chemical inactivation of the biological activity of DNA occurred during the manufacturing process.\n\nTaking into account the three requests described above, the product is considered by their regulatory organs (NRAs) and control laboratories (NLC), to be at an acceptable level of risk regarding the presence of DNA from the cell substrate, based on (a) and / or (b) and / or (c), when the data show that adequate levels of safety have been achieved.\nIn particular in the 2 batches of Priorix Tetra vaccine tested to date, point A. it does not occur because the quantity is about 140 times higher than that recommended by the FDA (in Briefing Document September 19, 2012: Vaccines and Related Biological Products Advisory Committee Meeting) and by the EMA, ie ≤ 10ng per dose; point b) does not occur because the DNA is high molecular weight (most> 10,000 bp, as can easily be verified using a simple agarose gel to control the quality of the DNA extracted from the vaccine), ie 50 times greater than the size recommended by the FDA (200bp or less). Finally, in the same vaccine, point c) does not occur because, containing attenuated viruses, a possible chemical inactivation of DNA, it would also inactivate viruses.\n\nPriorix Tetra lot comparison\nLot # 1 - A71CB205A Lot # 2 - A71CB256A DNA analysis\nTotal DNA extracted: 1.7 μg total per 0.5mL dose\nPerformed DNA-seq analysis using a metagenomic approach, suuntotaledi3 .830.074 of sequences produced\n\nPresent genomic DNA of:\nVaricella1 4%\nPollo 4% \nMan (MRC-5) 74% \nproteobacteria 1% \nRNA virus 0.01% not assigned \n5%DNA analysis\n\nTotal DNA extracted: 3.7 μg total per 0.5mL dose\nPerformed DNA-seq analysis using a metagenomic approach, suuntotaledi5 .836.297 of sequences produced.\n\nPresent genomic DNA of:\nVaricella1 4% \nMan (MRC-5) 8 8% (about 3.3 μg equivalent to about 300,000 fetal cells) 0.0003% \nRNA virus not assigned \n0.5%RNA analysis\n\nTotal RNA extracted:\nnot quantifiable with standard fluorimetric methods\n\nPerformed RNA-seq analysis using a metatrascritomic approach,\non a total of 10.445.038 of sequences produced.\nMeasure\n0.004% Mumps \n0.008% Rubella\n0.00007% Varicella\n5% other viruses \nabout 0.002% helminth \n0.6% P o l l o \n0. 2% for men \n87%\nnot allocated 5%\n\nRNA analysis\n\nTotal RNA extracted: 200ng per 0.5mL dose\nPerformed RNA-seq analysis using a metatrascritomic approach,\nout of a total of 6,171,266 of produced sequences.\nMeasles 0.004% \nMumps 0.008% \nRosolian nd% * \nVaricella 7%\nother viruses about 0.001%\nnematoda 1.50% \nProteobacteria 5.5%\nU or m o 6 8%\nnot allocated 6%\n* sequencing with 260.343.42 reads: 114 reads equal to 0.00004%\n\nMethods and results\n\nDNA and RNA extraction Priorix Tetra lot. A71CB205A\nThe lotA71CB205A lot was tried in June 2018.\nGenomic DNA extraction was carried out with the Maxwell® 16 Blood DNA Purification Kit sold by Promega and with the automatic extractor Maxwell® 16 IVD (Promega), following the manufacturer's protocol. RNA extraction was performed using the PureLinkTM Viral RNA / DNA Kit Mini Kit (Invitrogen) following the manufacturer's protocol.\n\nThe starting quantities used for the extractions are as follows (starting from a single vial of product):\n● DNA extraction: 125 μl on 500 μl total of suspension for injection\n● RNA extraction: 125 μl on 500 μl total of suspension for injection\n\nThe quantification and quality control of the extracted DNA and RNA was carried out with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).\nBelow is the result of the quantifications (ND = NanoDrop 1000; QB = Qubit 2.0; HS = dsDNA HS Assay Kit)\n\nThe measurement of DNA concentration with QuBit fluorometer showed that the lot A71CB205A contains a quantity of gDNA of 1.7 μg total per dose of 0.5mL, calculated as follows:\n9.41ng / μl (concentration determined at QuBit) x 45 (final resuspension volume of DNA after extraction, expressed in microliters) x 4 (the starting volume subjected to the extraction procedure is 1/4 of the volume of the dose contained in the whole vial equal to 0.5mL).\n\nDNA and RNA extraction Priorix Tetra lot. A71CB256A\nLot A71CB206A was tried in December 2018. Some improvements were made in the procedure, such as:\n1. Use of a higher volume of injectable starting solution in order to increase the amount of extracted RNA (in the extraction from the previous batch the amount of RNA obtained was below the detection threshold with fluorimeter);\n2. Performed an electrophoresis in the pulsed field, to get a greater detail on the size of the whole genomic DNA present in the sample;\n3. Used a measurement mode for the most sensitive RNA (Agilent RNA 6000 Pico Kit on Agilent Bioanalyzer).\n\nGenomic DNA extraction was carried out with the Maxwell® 16 Blood DNA Purification Kit sold by Promega and with the automatic extractor Maxwell® 16 IVD (Promega), following the manufacturer's protocol. RNA extraction was performed using the PureLinkTM Viral RNA / DNA Kit Mini Kit (Invitrogen) following the manufacturer's protocol.\n\nThe starting quantities used for the extractions are as follows, starting from two vials of the product of the same lot:\n● DNA extraction: 300μl on 500μl suspension for injection (ampoule 1)\n● RNA extraction: the entire volume of powder in a vial was resuspended in 200 μl of physiological saline supplied in the package, instead of 500 μl, and the entire volume was used for\nRNA extraction (vial 2).\n\nThe quantification and quality control of the extracted DNA was carried out respectively with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and with the spectrophotometer N anoDrop1000 (ThermoFisherScientific, Waltham, USA).\nFollowing is the result of DNA quantifications (ND = NanoDrop 1000; QB = Qubit 2.0; HS = dsDNA HS Assay Kit)\n\nThe measurement of DNA concentration with the QuBit fluorometer showed that the lot A71CB256A contains an amount of gDNA of 3.7 μg total per dose of 0.5mL, calculated as follows:\n40.8 ng / μl (concentration determined at QuBit) x 55 (final resuspension volume of DNA after extraction expressed in microliters) x 5/3 (the initial volume subjected to the extraction procedure is 300 μl on 500 μl of suspension) .\nElectrophoresis in pulsed field (PFGE, 5-80Kb, stroke 14h in TBE 0.5x, 80V) of DNA extracted from lot A71CB256A, made visible thanks to SybrGreen fluorescent interlayer, highlighted the presence of a wide 'strip' of DNA genomic that reaches up to very high molecular weights, but with a significant amount of DNA in the 20-60Kbp range. In particular in the photo below, the sample ID1205_PT _raw is the lysate of the material contained in the vaccine before DNA purification, while PT_gDNA is the genomic DNA after extraction; Mid, Low and Lambda are 3 commercial molecular weight markers.\nThe quantification and quality control of the extracted RNA was performed on Agilent 2100 Bioanalyzer using the A gilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA). Below are the concentration values, the RNA (Integrity Number) measured at Bionalyzer:\n\nThe amount of RNA contained in the lot A71CB256A vaccine vial was found to be about 200ng.\nThe RIN of 8 indicates an RNA of excellent quality and an intact eukaryotic RNA, being present both the 18S and 28S peaks typical of eukaryotic RNA:\nPreparation of the DNA-seq library with Illumina technology\nThe Ovation® Ultralow System V4 1-96 kit (Nugen, San Carlos, CA) was used to prepare the libraries, following the manufacturer's indications, starting from 10ng of genomic DNA. The final libraries were quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and tested for quality with the Caliper GX system (PerkinElmer, Waltham, MA) for the lot A71CB205A and Agilent 2100 Bioanalyzer, DNA High Sensitivity Analysis kit (Agilent technologies, Santa Clara , CA) for the lot A71CB256A. \n\nFollowing are the traces for the two libraries obtained:\nPreparation of the RNA-seq library with Illumina technology\nFor the preparation of the RNA-seq libraries the Ovation® RNA-Seq System V2 kit (Nugen, San Carlos, CA) was used to prepare the cDNA and the Ovation® Ultralow System V4 1-96 kit to prepare the library starting from 10ng of cDNA. The final libraries were quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and tested for quality with the Caliper GX system (PerkinElmer, Waltham, MA) for the lot A71CB205A and Agilent 2100 Bioanalyzer, DNA High Sensitivity Analysis kit (Agilent technologies, Santa Clara, CA) for the lot A71CB256A. \n\nFollowing are the traces for the two libraries obtained:\n\nIn order to validate the workflow from the preparation of the library to the analysis of the data, an ATCC standard (mix of genomic DNA with a known composition, 20 Strain Staggered Mix Genomic Material, ATCC® MSA-1003TM) was used to construct a library with the kit Ovation®UltralowSystemV4 1-96 starting from 10ng of DNA. Following is the path to the Bioanalyzer of the library obtained:\n\nSequencing\n\nThe libraries have been sequenced on Illumina HiSeq2500 instrument in 125bp 'paired-end' mode, according to Illumina standard indications. Version 1.8.2 of the Illumina CASAVA pipeline was used to process raw sequences.\n\nBioinformatic analysis\n\nCleaning of sequences (trimming)\nThe sequences of the adapters (ie 'artificial' oligonucleotide sequences that are introduced during the Illumina library preparation) and the low quality read DNA bases have been removed using the ERNE1 and Cutadapt2 software.\nIdentification of organisms of origin of DNA and cDNA sequences Metagenomic analysis was performed using the Kraken3 software on the 'Human-Virus-Bacteria_25mer' database\n(h ttps: //ccb.jhu.edu/software/kraken/).\n\nKraken is a classifier that assigns taxonomic labels to short DNA readings. It does this by examining the k-mers in a reading and querying a database with those k-mers.\n\nBibliographic references\n1. Del Fabbro, C et al. 2013 An evaluation of readings on Illumina NGS data analysis. Del Smith C, Scalabrin S, Morgante M, Giorgi FM. PLoS One. 2013 Dec 23; 8 (12): e85024. doi: 10.1371 / journal.pone.0085024. eCollection 2013\n2. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, [S.l.], 17 (1): 10-12 (2011). ISSN 2226-6089. Date accessed: 02 Apr. 2015. doi: http: //dx.doi.org/10.14806/ej.17.1.200 paper\n3. Wood and Salzberg. Kraken: ultrafastmetagenomicsequence classification using exact alignments Genome Biology 2014, 15: R46\n\nFrom the comparison of these two vaccines it is possible to highlight the following critical issues:\nThe Priorix Tetra is a vaccine with a high amount of contaminating foreign DNA of which on average 80% is human, therefore coming from MRC-5 cells; in Lot # 1 there is also 4% of DNA coming from embryo chicken cells; the remaining 20% belongs to viruses (retroviruses, infectious viruses, and carcinogens, phages) and adventitious microorganisms such as bacteria, and worms; in the Priorix Tetra vaccine the human genomic DNA is high molecular weight (> 10.000bp) and the total sequential coverage of the entire reference human genome (HG-19) shows that it is the entire genome of the fetal cells used for the culture of vaccine viruses to be present and not just portions of it.\n\nFrom the EMA's response to our question6 on the limits imposed on residues of foreign genetic material in vaccines, it appears that in fact there are no limits for each vaccine but only for some, reported in the monographs of the product; The maximum limit envisaged ranges from 10 pg 7 to 10 ng, based on the theoretical calculation of the possibility of foreign genomic DNA to cause oncogenic mutations.\n\nIt is noteworthy that regulatory authorities do not require that these contaminations be tested in the final product, but only in the initial preparation phase, and that for the attenuated virus vaccines the purification of these contaminations is a critical step8. The EMA has not provided specific studies on the dangers of fetal residual DNA, which allow to assess the risk to human health of these contaminations, so this limit remains arbitrary today.\n\nIn Lot # 2 of Priorix Tetra, fetal DNA is about 325 times higher than the maximum limit of 10 ng and 325,000 times higher than the minimum limit of 10 pg.\n\nOn the question of contaminating human DNA, the World Health Institute in an official 2011 document entitled 'Recommendations for the Evaluation of Animal Cells and Subsidies for the Production of Biological Products and Cells' argues that what is necessary to take into consideration with respect to rcDNA (residual cell DNA) in vaccines is:\n\nA. a reduction in the amount of contaminating DNA during the manufacturing process;\n\nB. a reduction in the size of the contaminating DNA during the manufacturing process;\n\nC. a chemical inactivation of the biological activity of DNA occurred during the manufacturing process.\n\nTaking into account the three requests described above, the product is considered by their regulatory organs (NRAs) and control laboratories (NLC), to be at an acceptable level of risk regarding the presence of DNA from the cell substrate, based on (a) and / or (b) and / or (c), when the data show that adequate levels of safety have been achieved.\n\nIn particular in the 2 batches of Priorix Tetra vaccine tested to date, point A. it does not occur because the quantity is about 140 times higher than that recommended by the FDA (in Briefing Document September 19, 2012: Vaccines and Related Biological Products Advisory Committee Meeting) and the EMA, ie ≤ 10ng per dose; point b) does not occur because the DNA is high molecular weight (most> 10,000 bp, as can easily be verified using a simple agarose gel to control the quality of the DNA extracted from the vaccine), ie 50 times greater than the size recommended by the FDA (200bp or less). Finally, in the same vaccine, point c) does not occur because, containing attenuated viruses, a possible chemical inactivation of DNA, it would also inactivate viruses.\n\nAnalysis of Genetic Variants\n\nWith Next Generation Sequencing technology it is possible to reconstruct the entire sequence of viral DNA and RNA genomes and bacterial genomes present in the sample and compare it with the reference genomes present in public databases. The technology can therefore also allow to monitor in time how and if the sequence of a viral or bacterial genome changes during the production procedure of a vaccine.\n\nThe result of the variants call (single nucleotide and small insertions / deletions) compared to the reference strains found in public databases (NCBI, National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) carried out in the vaccine samples containing live attenuated virus or bacteria gave the following results:\n\nSample 1. - Priorix Tetra\n\n1. The genome of the measles virus contained in the vaccine is identical to the sequence of the Edmonston Swartz strain deposited in the databases having accession number AF266291.1.\nThe number of variants detected was in fact equal to 0;\n\n2. The genome of the mumps virus contained in the vaccine showed a single mutation compared to the Jeryl-Lynn viral strain present in public databases with the accession number AF338106.1;\n\n3. The genome of the rubella virus has not been detected;\n\n4. The genome of the varicella virus contained in the vaccine has shown four mutations compared to the Human herpesvirus 3\npresent in public databases with access number AB097932.1;\n\nThe sequence of viral antigens / genomes is a strictly confidential data that is not provided by the EMA. There are no guidelines that regulate the analysis of genetic mutations and the study of effects on human health.\n\nThe high frequency of genetic mutations in viruses and bacteria, as well as in the DNA of cell lines in culture, is a problem of great importance with regard to safety, as it is not known how any variants found can modify the infectious capacity and the stimulation of the immune system towards autoimmune reactions.\n\nFor example, it is suggested that Efsa now requires the genomic characterization of probiotic strains for human / animal use and subsequently the demonstration of the coincidence, over time, of the sequence of the microorganism compared to that declared, while in the case of vaccines, as Vivotif, are tolerated as many as 154 genetic variants compared to that stated in the data sheet and present in public databases as a reference vaccine strain.\n\nThe presence of genetic variants in vaccine samples compared to the strains declared can be considered in our opinion a non-compliance of drugs.\n\nDeepening\n\nIn the recent prepublished article on F1000research \"Do you cov me? NGS technology has been used to analyze biological matrices of different types, including two batches of measles-mumps-rubella-varicella vaccine. PRIORIX TETRA (GlaxoSmithKline SpA), with the aim of demonstrating how, even from low-coverage NGS sequencing (ie of a few hundred thousand sequence fragments to 1 million), it is possible to characterize the biological component in a complex matrix. The next-generation sequencing was already used on vaccine samples in the publication \"Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis\" to demonstrate the coincidence between the genome of the vaccine virus of mumps and the virus found in the brain tissue of an 18-month-old child with SCID who died of encephalitis.\n\nIn particular, in the pre-published article on F1000research, it is observed that about 80% of the sequences obtained with NGS technology on the two vaccine samples, consisting of human DNA, as impurity present in the working process; the total amount of DNA extraneous is about 2 micrograms, coming from the human fetal cell line MRC-5 used to grow rubella and varicella viruses). The metagenomic analysis performed on these two samples highlights the potential presence of human DNA in all vaccines containing viruses grown in human fetal lines, also already verified with technology other than NGS also by Dr. Deisher in \"Epidemiologic and Molecular Relationship Between Vaccine Manufacture and Autism Spectrum Disorder Prevalence \".\n\nDr. Theresa Deisher's group in the article \"Insertional mutagenesis and autoimmunity induced disease caused by human fetal and retroviral residual toxins in vaccines\" states that the levels of residual DNA in MPR, chickenpox and hepatitis A vaccines available in the United States are abundant the limit set in the current WHO DNA Guideline from immortalized cell lines of 10 ng per dose of vaccine. Although the EMA guideline does not provide for maximum limits for the fetal DNA remaining in the vaccines, Prof. Deisher's group has also taken as reference the maximum dose of 10 ng as a consequence of the fact that the short fragments of fetal DNA present in the vaccines present the ability to integrate into the host DNA and can lead to mutagenesis and / or genomic instability as well as an autoimmune response. Furthermore, in some varicella and MPR vaccines, the presence of fragments of endogenous human retrovirus K (HERVK) has been found, which can be re-activated and can facilitate the integration of free DNA into the host genome.\n\nAs stated by Dr. Deisher, the danger of retroviral fragments and residual human diploid DNA has not yet been studied in the recipients of the vaccine, although the scientific literature clearly demonstrates the high probability of the dangers of autoimmune and / or insertional mutagenesis due to presence of these residues, and this is a risk to human health that undoubtedly requires serious scientific and epidemiological research.\nThese results are integrated with the new data on the analysis of a new sample of Priorix tetra and the reanalysis of the previous ones, as a non-conformity has emerged concerning the vaccine antigens, ie the dubious presence of the rubella antigen. From the in-depth analysis of the three lots it was confirmed that the rubella antigen is not present, because the number of copies per sample is totally negligible to have an immunostimulatory effect.\n\nThis more in-depth investigation has made it possible to detect the presence of adventitious DNA and RNA, ie from viruses, bacteria, fungi and helminths in quantities below the limit of detection of the instrument, and therefore in residual quantities. However, it should be emphasized that the average total amount of total DNA varies from 1.9 to 3.7 micrograms, of which about 80% comes from human fetal DNA and the remaining 20% from embryo chicken DNA and adventitious genetic material, then in quantity cumulative non-residual.\n \nIt should be noted that in the various lots a considerable variability of the adventitious contaminants was noted, but overall the following categories of adventitious microorganisms were found:\n\n■ Bacteria \n■ Proteobacteria\n■ Worms \n■ Platyhelminthes \n■ Nematoda\n■ dsDNA viruses \n■ Varicella virus\n■ ssRNA viruses \n■ Rubella, mumps and measles virus\n■ Other viruses including Influenza A, Cupixi mammarenavirus, Pneumoviridae, Jamestown Canyon, Hepacivirus C, Kobuvirus Enterovirus, Porcine reproductive and respiratory syndrome virus\n■ Retrovirus\n■ human and avian endogenous retroviruses\n■ avian viruses\n■ human and monkey immunodeficiency viruses \n■ murine viruses\n■ Infectious horse anemia virus \n■ Lymphoproliferative disease virus \n■ Rous sarcoma virus\n■ Other viruses \n■ Human alphaherpesvirus 3 \n■ Hepatitis b virus\n■ Yeast virus\n\nhttps://drive.google.com/file/d/1mufQ9Ueoph4T4BufJ71M6v95KvOfhXAw/view ",
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