Vaccines-Control and Investigations: Micro and Nanocontamination

Vaccines are one of the most notable inventions meant to protect people from infectious diseases. The practice of variolation is century-old and is mentioned in Chinese and Indian documents dated around 1000 A.D. Over time, variolation has been replaced by vaccination, vaccines have been enhanced as to technology, and the vaccination practice is now standardized worldwide.Side effects have always been reported but in the latest years it seems that they have increased in number and seriousness, particularly in children as the American Academy of pediatrics reports [1,2]. For instance, the diphtheria-tetanus-pertussis (DTaP) vaccine was linked to cases of sudden infant death syndrome (SIDS) [3]; measles-mumps-rubella vaccine with autism [4,5]; multiple immunizations with immune disorders [6]; hepatitis B vaccines with multiple sclerosis, etc. The notice of Tripedia DTaP by Sanofi Pasteur reports “Adverse events reported during post-approval use of Tripedia vaccine include idiopathic thrombocytopenic purpura, SIDS, anaphylactic reaction, cellulitis, autism, convulsion/grand mal convulsion, encephalopathia, hypotonia, neuropathy, somnolence and apnea”. The epidemiological studies carried out did not show a clear evidence of those associations, even if in 2011 the National Academy of Medicine (formerly, IOM) admitted: “Vaccines are not free from side effects, or adverse effects”[7]. Specific researches on components of the vaccines like adjuvants (in most instances, Aluminum salts) are already indicated as possible responsible of neurological symptoms [8-10] and in some cases, in-vivo tests and epidemiological studies demonstrated a possible correlation with neurological diseases [10,11]. Neurological damages induced in patients under hemodialysis treated with water containing Aluminum are reported in literature [12]. Recently, with the worldwide-adopted vaccines against Human Papillomavirus (HPV), the debate was reawaken due to some adverse effects reported by some young subjects. Specific studies communicated the existence of symptoms related to never-described-before syndromes developed after the vaccine was administered. For instance, Complex Regional Pain Syndrome (CRPS), Postural Orthostatic Tachycardia Syndrome (POTS), and Chronic Fatigue Syndrome (CFS) [13]. The side-effects that can arise within a relatively short time can be local or systemic. Pain at the site of injection, swelling and uncontrollable movement of the hands (though this last symptom can also be considered systemic) are described. Among the systemic effects, fever, headache, irritability, epileptic seizures, temporary speech loss, lower limbs dysaesthesia and paresis, hot flashes, sleep disorders, hypersensitivity reactions, muscle pain, recurrent syncope, constant hunger, significant gait impairment, incapacity to maintain the orthostatic posture are reported [14]. It is a matter of fact that every day millions of vaccine doses are administered and nothing notable happens, but it is also irrefutable that, regardless of the amount of side effects that are not recorded and the percentage of which remains in fact unknown, in a limited number cases something wrong occurs. No satisfactory explanation or, in many cases, no explanation at all has been given and it seems that those adverse effects happen on a random and stochastic basis. List of the vaccines according to their manufacturers with the chemical composition of the debris identified in each sample. The elements most represented are reported. N Company Name Alluminum Elements Identified 1 Allergopharma - Germany Allergoid yes Al 2 Aventis Pasteur MSD Lyon - Francie Typhim Vi no BrKP, PbSi, FeCr, PbClSiTi 3 Baxter AG Tetabulin no SiMg, Fe, SiTiAl, SBa, Zn 4 Berna Biotech Vivotif Berna no FeAl, ZrAlHf, SrAl, BiAlCl 5 Berna Biotech Inflexal V no CuSnPbZn, Fe, CaSiAl, SiAl, NaPZn, ZnP, AlSiTi 6 Chiron Anatetall Al(OH)3 FeAl, SZnBaAl 7 Chiron Morupar no / 8 GlaxoSmithKline- Belgium Mencevax ACWY no FeCrNi, ZrAl, FeCrNiZrAlSi 9 GlaxoSmithKline Infanrix Al(OH)3 Al, AlTi, AlSi 10 GlaxoSmithKline Biologicals Infanrix hexa Al(OH)3 SBa, FeCu, SiAl, FeSi, CaMgSi, AlCaSi, Ti, Au, SCa, SiAlFeSnCuCrZn, CaAlSi 11 GlaxoSmithKline Biologicals Infanrix hexa Al(OH)3 ,AlPO4. 2H2O W, FeCrNi, Ti 12 GlaxoSmithKline Typherix no Ti, TiW, AlSiTiWCr, SBa, W, SiAl, AlSiTi 13 GlaxoSmithKline Priorix no WCa, WFeCu, SiAl, SiMg, PbFe, Ti, WNiFe 14 GlaxoSmithKline Engerix-B no Al (precipitates) 15 GlaxoSmithKline Varilrix no FeZn, FeSi, AlSiFe, SiAlTiFe, MgSi, Ti, Zr, Bi 16 GlaxoSmithKline Fluarix no AlCu, Fe, AlBi, Si, SiZn, AlCuFe, SiMg, SBa, AlCuBi, FeCrNi, SPZn 17 GlaxoSmithKline Biologicals Cervarix Al(OH)3 AlSi,FeAl, SiMg,CaSiAl, CaZn, FeAlSi, FeCr, CuSnPb 18 Novartis Vaccines and Diagnostics Anatetall Al(OH)3 Al, FeCrNi, AlCr, AlFe, BaS, ZnAl 19 Novartis Vaccines and Diagnostics Dif-Tet-All Al(OH)3 Fe,SBa, SiSBa, AlZnCu, AlZnFeCr 20 Novartis Vaccines and Diagnostics Menjugate kit Al(OH)3 SiAl,Ti,FeZn, Fe, Sb, SiAlFeTi, W, Zr 21 Novartis Vaccines and Diagnostics Focetria no Fe, FeCrNiCu, FeCrNi, SiFeCrNi, Cr, SiAlFe, AlSiTiFe, AlSi, SiMgFe, Si, FeZn 22 Novartis Agrippal S1 no Ca, Fe, SBa, SBaZn, Cr, Si, Pb, Bi, e FeSiAlCr, SiAlSBaFe, CaAlSi, Zn, CeFeTiNi, FeCrNi 23 Novartis Vaccines and Diagnostics Agrippal S1 no SiAlK, Si, SiMgFe, CaSiAl, SBaZn 24 Novartis vaccines Agrippal no Cr, Ca, SiCaAl, ZrSi, SBa, CuZn, SCa 25 Novartis Vaccines and Diagnostics S Fluad no CaSiAl, FeSiTi,SiMgAlFe, SBa 26 Novartis Vaccines and Diagnostics Menveo no CaSiAl, SiAlFe, FeCrNi, Fe, Al, SBa 27 Pfizer Prenevar 13 no FeCr 28 Pfizer Prevenar 13 no W, CaAlSi, Al, CaSiAlFe, FeS, FeCr, FeCrNi, Fe, , CaP, FeTiMn, Ba, SiMgAlFe 29 Pfizer Meningitec - ctrl no Cr, Si 30 Pfizer Meningitec - ctrl no FeCrNi, W 31 Pfizer Meningitec no CaSiAl, CaSi, SiAlFeTi, FeCrNi, W, Fe, Pb 32 Pfizer Meningitec no Cr (precipitates), Ca, AlSi 33 Pfizer Meningitec no W, SiCa, CaSi, Pb, FeCrNi, Cr 34 Wyeth Pharmaceutical - UK Meningitec no SiAlFe, SiAlTi, SiMgFe, W, Fe, Zr, Pb, Ca, Zn, FeCrNi 35 Sanofi Pasteur MSD-France Vaxigrip no Fe, FeCrNi, SiAlFe, AlSi, SiAlFeCr 36 Sanofi Pasteur MSD Stamaril Pasteur no CaSiAl, AlSi, Fe, SiMgFe, SiMgAlFe, CrSiFeCr, CrSiCuFe 37 Sanofi Pasteur MSD Gardasil AlPO4. 2H2O AlCuFe, PbBi, Pb, Bi, Fe 38 Sanifi Pasteur MSD Gardasil AlPO4. 2H2O CaAlSi, AlSi, SiMgFe, Al,Fe, AlCuFe, FeSiAl, BiBaS, Ti, TiAlSi 39 Sanofi Pasteur Vaxigrip no Ca, CrFe, FeCrNi, CaSZn, CaSiAlTiFe, Ag, Fe 40 Sanofi Pasteur Vaxigrip no SiMgFe, CaSiAl, AlSiFe, AlSi,FeCr, FeZn, Fe 41 Sanofi Pasteur MSD Repevax AlPO4 .2H2O Bi, Fe, AlSiFe, SiMg, SBa, Ca 42 Sanofi Pasteur MSD S Repevax AlPO4.2H2O Ti, Br, AuCuZn, Ca, SiZn, SiAuAgCu, SiMgFe,FeCrNi. AlSiMgTiMnCrFe, SiFeCrNi, FeAl 43 Sanofi Pasteur MSD M-M-R vaxPro no Si, SiFeCrNi, FeCrNi,FeNi, Fe, SCa, AlSiCa, CaAlSiFeV, SBa, Pt, PtAgBiFeCr 44 Virbac S.A. - Carros - France Feligen CRP no Ca,SiAl N Name Brand Name, Country of Distribution Description Production Batch, Expiry Date 1 Vivotif Berna Berna Biotech SA, Italy Anti-Thyphoid Vaccine (Live), group Ty21a 3000336 [2004] 2 Typhim Vi Aventis Pasteur MSD, Italy Anti-Salmonella typhi Vaccine U1510-2 [2004] 3 Typherix GlaxoSmithKline S.p.a., Italy Anti-Thypoid Vaccine (polysaccharide Vi) ATYPB061BB [2009] 4 Anatetall Chiron (now Novartis) Italy Adsorbed anti-Tetanus Vaccine 030106 [2004] 5 Anatetall Novartis Vaccines and Diagnostics, Italy Adsorbed anti-Tetanus Vaccine 060510 [2009] 6 Tetabulin Baxter AG, Italy Adsorbed anti-Tetanus Vaccine VNG2G006A [2009] 7 Dif-Tet-All Novartis Vaccines and Diagnostics, Italy Adsorbed anti-Tetanus and diphtheria Vaccine 070501 [2009] 8 Infanrix GlaxoSmithKline S.p.a., Italy Anti-Diphtheria, tetanus and pertussis vaccine AC14B071AJ [2009] 9 Infanrix hexa GlaxoSmithKline Biologicals s, Italy Anti-diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis and disease caused by Haemophilus influenzae type b A21CC512A [ 2017] 10 Infanrix hexa GlaxoSmithKline Biologicals s. a. France Anti-diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis and disease caused by Haemophilus influenzae type b A21CC421A [ 2017] 11 M-M-R vaxPro Sanofi Pasteur MSD, Italy M-M-R vaxPro (measles, mumps, and rubella) analyzed in Cambridge L012437 [ 2017] 12 Repevax Sanofi Pasteur MSD, France Anti-diphtheria-tetanus-pertussis-polio-vaccine L0362-1 [2017] 13 Repevax Sanofi Pasteur MSD SNC France Anti-diphtheria-tetanus-pertussis-polio-vaccine L0033-1 [2016] 14 Priorix GlaxoSmithKline S.p.a., Italy Anti--measles-mumps, and rubella (MMR) vaccine A69CB550A [2009] 15 Morupar Chiron (now Novartis, ), Italy Anti-measles- mumps, and rubella (MMR) vaccine 7601 [2004] 16 Varilrix GlaxoSmithKline S.p.a., Italy Anti-Chicken pox vaccine (group OKA) A70CA567A [2009] 17 Stamaril Pasteur Sanofi Pasteur MSD, Italy anti-yellow fever vaccine A5329-6 [2009] 18 Allergoid-Adsorbat 6-Graser Starke B. Allergopharma, Germany Antiallergic vaccine Ch-B.:30005999-B [2006] 19 Engerix-B GlaxoSmithKline S.p.a., Italy Adsorbed anti-hepatitis B vaccine AHBVB468BD [2009] 20 Prenevar 13 Pfizer, Italy Antipneumococcal vaccine G79324 [2013] 21 Prevenar 13 Pfizer, France Antipneumococcal vaccine N27430 [ 2018] 22 Mencevax Acwy GlaxoSmithKline, Italy anti-Neisseria meningococcal group A, C, W135 and Y vaccine N402A47B 12 [2004] 23 Meningitec Pfizer, Italy (group C 10) (adsorbed on Al-Phosphate) H92709 [2015] 24 Meningitec Pfizer-Italy Anti-meningococcus (group C 10) vaccine (adsorbed on Al-Phosphate) H20500 [2014] 25 Meningitec Pfizer-Italy Anti-meningococcus vaccine sequestred by Procura della Repubblica G76673 [2014] 26 Meningitec Pfizer-Italy Anti-meningococcus vaccine sequestred by Procura della Repubblica H99459 [2015] 27 Meningitec Pfizer-Italy Anti-meningococcus vaccine sequestred by Procura della Repubblica H52269 [2015] 28 Menjugate Novartis Vaccines and Diagnostics Anti-meningococcus group C YA0163AB [2010] 29 Menveo Novartis Vaccines and Diagnostics Antimeningococcus groups A, C, W135, Y A15083 [2017] 30 Meningitec Wyeth Pharmaceutical - France Anti-meningococcus group C vaccine E83920 [2011] 31 Inflexal V Berna Biotech Anti-flu vaccine 2008/2009 3001463-01 [2009] 32 Vaxigrip Sanofi Pasteur MSD Anti-flu vaccine 2008/2009 D9703-1 [2009] 33 Vaxigrip Sanofi Pasteur Anti-flu vaccine 2012/2013 J8401-1 [2013] 34 Vaxigrip Sanofi Pasteur, Italy Anti-flu vaccine, with inactivated and split virus M7319-1 [2016] 35 Focetria Novartis Vaccines and Diagnostics Anti-pandemic flu H1N1 vaccine 0902401 [2010] 36 Agrippal Novartis Anti-flu vaccine 2012/2013 127002A [2013] 37 Agrippal Novartis vaccines, Italy Anti-flu vaccine with inactivated and split virus 2015/2016 - 152803 [2016] 38 Agrippal S1 Novartis Vaccines and Diagnostics Anti-flu inactivated/superficial antigene v - 2014/2015 147302A [2015] 39 Fluarix GlaxoSmithKline - GSK Anti-flu vaccine 2013 AFLUA789AA [2014] 40 Fluad Novartis Vaccines and Diagnostics Anti-flu inactivated/superficial antigene vaccine - 2014/2015 142502 [2015] 41 Gardasil Sanofi Pasteur MSD, Italy Anti-HPV types 6,11,16,18 vaccine NP01250 [2012] 42 Gardasil Sanofi Pasteur MSD, Italy Anti-HPV (types 6,11,16,18) vaccine K023804 [2016] 43 Cervarix GlaxoSmithKline Biological, Italy Anti-HPV (type 16,18) AHPVA238AX [2017] 44 Feligen CRP Virbac S.A. - Carros - Italy anti-panleucopenia, infectious rhinotracheitis and infections by Calcivirus, veterinary Vaccine for cats 3R4R [2013] Some vaccines, in fact a minority, are meant to deal with a single bacterium or virus, while others are multi-valent. The list of vaccines we analyzed may contain repeated names, because we considered different batches and years of production of the same vaccine: the ones against influenza in particular. The study was aimed at verifying a possible physical contamination. To do that, we performed a new kind of investigation based on observations under a Field Emission Gun Environmental Electron Scanning Microscope (FEG-ESEM, Quanta 200, FEI, The Netherlands) equipped with the X-ray microprobe of an Energy Dispersive Spectroscope (EDS, EDAX, Mahwah, NJ, USA) to detect the possible presence of inorganic, particulate contaminants and identify their chemical composition. A drop of about 20 microliter of vaccine is released from the syringe on a 25-mm-diameter cellulose filter (Millipore, USA), inside a flow cabinet. The filter is then deposited on an Aluminum stub covered with an adhesive carbon disc. The sample is immediately put inside a clean box in order to avoid any contamination and the box is re-opened only for the sample to be inserted inside the FEG-ESEM chamber. We selected that particular type of microscope as it allows to analyse watery and oily samples in low vacuum (from 10 to 130 Pa) at a high sensitivity. When the water and saline the vaccine contains are evaporated, the biological/physical components emerge on the filter and it is then possible to observe them. This type of microscope (low-vacuum observations) prevents the possible sample contamination and the creation of artefacts. The observations are made with different sensors (SE: secondary-electron sensor and BSE: backscattered-electron sensor), and are performed at a pressure of 8.9 e-1 mbar, at energies ranging from 10 to 30kV to detect the particulate matter’s size, morphology and its elemental composition. The method identifies clearly inorganic bodies with a higher atomic density (looking whiter) than the biological substrate. So, organic entities are visible and easy to distinguish from inorganic ones. The method cannot distinguish between proteins and organic adjuvants (e.g. squalene, glutamate, proteins, etc.) or viruses, bacteria, bacteria’s DNA, endo-toxins and bacteria’s waste, but their comparatively low atomic density allows us to identify these entities as organic matter. In some vaccines, the organic matter contains white-looking debris named aggregates, while a high concentration or inorganic debris is called a cluster. Single inorganic particles or organic-inorganic aggregates are identified, evaluated and counted. The counting procedure is repeated three times by three different operators, with an error lower than 10%. When a layer of salts (Sodium chloride or Aluminum) is detected, we record the situation but we do not do body count.