viral dna bound to aluminum may mediate hpv vaccination ...8 . five gardasil vaccine samples from...

Viral DNA bound to aluminum may mediate HPV vaccination side effects

Sin Hang Lee MD FRCP(C) FCAP

Director

Milford Molecular Diagnostics Laboratory

Milford Connecticut 06460

USA

Conflicts of interest

This work was performed when Dr Lee was a pathologist at Milford

Hospital and sponsored by SANEVAX Inc for a future payment not to

exceed one US dollar

1

Pathology is a specialty of Medicine

bull Why or how people die

bull Why or how people get sick

bull Analyze patient samples for diagnosis

bull Molecular pathology

Sanger sequencing DNA analysis for

diagnosis of HPV Chlamydia gonorrhea

Lyme disease Ebola and breast cancer gene

mutations ndash

Precision Molecular Personalized Medicine

2

Evidence-based Diagnosis of Left Bundle Branch Block Seizure and HPV-18

by EKG EEG and DNA Sequencing

4

1 Lee S H Vigliotti VS Vigliotti JS and Pappu S Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories Infect Agent Cancer 2007 211

2 Lee S H Vigliotti VS and Pappu S DNA Sequencing Validation of Chlamydia trachomatis and Neisseria gonorrhoeae Nucleic Acid Tests Am J Clin Pathol 2008129852-859

3 Lee SH Vigliotti VS Pappu S Human papillomavirus (HPV) infection among women in a representative rural and suburban population of the United States Inter J Gyn Ob 2009 105210-214

4 Lee SH Vigliotti VS and Pappu S Molecular tests for human papillomavirus (HPV) Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cytology specimen BMC Womenrsquos Health 2009 98

5 Lee S H Vigliotti VS Vigliotti JS and Pappu S Validation of human papillomavirus genotyping by signature DNA sequence analysis BMC Clin Pathol 2009 93

6 Lee SH Vigliotti VS and Pappu S Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens J Clin Path 201063235-239

7 Lee S H Vigliotti VS Vigliotti JS Jones W and Pappu S Increased Sensitivity and Specificity of Borrelia burgdorferi 16S Ribosomal DNA Detection Am J Clin Path 2010 133569-576

8 Lee SH From human papillomavirus to cervical cancer Obstet Gynecol 2010 1161221-1222 9 Lee S H Vigliotti VS Vigliotti JS Jones W Williams J Walshon J Early Lyme disease with

spirochetemia ndash diagnosed by DNA sequencing BMC Res Notes 2010 Nov 1 3273 10 Lee SH Chapter 5 in ldquoGuidelines for the use of molecular tests for the detection and genotyping of human

papilloma virus from clinical specimensrdquo Methods Mol Biol 2012 90365-101

11 Lee SH Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum

adjuvant in the HPV vaccine Gardasilreg J Inorg Biochem 2012 11785ndash92 12 Lee SH Detection of human papillomavirus L1 gene DNA fragments in postmortem blood and

spleen after Gardasilreg vaccination-A case report Advances in Bioscience and Biotechnology 2012 3 1214-1224

13 Lee SH Topological conformational changes of human papillomavirus (HPV) DNA bound to an insoluble aluminum salt ndash a study by low temperature PCR Advances in Biological Chemistry 2013 3 76-85

14 Hong G Lee SH Ge S Zhou S A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification International Journal of Molecular Sciences 2013 1412853-12862

15 Lee SH Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasilreg Curr Med Chem 2014 21932-940

16 Lee SH Vigliotti JS Vigliotti VS Jones W Shearer DM Detection of Borreliae in Archived Sera from Patients with Clinically Suspect Lyme Disease International Journal of Molecular Sciences 2014 154284-4298

17 Lee SH Vigliotti JS Vigliotti VS Jones W Moorcroft TA Lantsman K DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infections International Journal of Molecular Sciences 2014 1511364-11386

18 Lee SH Vigliotti JS Vigliotti VS Jones W From human papillomavirus (HPV) detection to cervical cancer prevention in clinical practice Cancers (Basel) 2014 6(4)2072-2099

19 Lee SH Zhou S Zhou T Hong G Sanger Sequencing for BRCA1 c68_69del BRCA1 c5266dup and BRCA2 c5946del Mutation Screen on Pap Smear Cytology Samples International Journal of Molecular Sciences 2016 17(2)229

20 Lee SH Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes a case report International Medical Case Reports Journal 2016 (in press)

Gardasil-related heart disease

bull FDA reminder 15-minute Observation Period Needed After

Vaccination ndash to prevent syncope

bull Postural Orthostatic Tachycardia Syndrome after Gardasil

vaccination is widely reported- POTS

bull A significant number of healthy active teenagers died

unexpectedly and suddenly (in sleep in bathtub under the

shower) after Gardasil vaccination- sudden deaths

bull At least one case of myocardial infarction in a 14-year old boy

closely related to Gardasil vaccination was filed in Federal

Court MI

5

Report of HPV DNA in blood of a 13-year old girl who developed

juvenile rheumatoid arthritis after Gardasil vaccination (2011)

DiMario FJ Jr Hajjar M Ciesielski T A 16-year-old girl with bilateral visual loss and

left hemiparesis following an immunization against human papilloma virus J Child

Neurol 2010 25321-327

We report the case of a 16-year-old girl who

suffered an acute and sustained onset of

bilateral visual loss and transient left

hemiparesis following an immunization against

human papilloma virus who was found to have

both a tumefactive demyelinating lesion and

chiasmal neuritis as part of a presentation of

acute demyelinating encephalomyelitis

7

Table 1 ndash Gardasilreg lot numbers countries of origin and HPV L1 gene DNA found

J Inorg Biochem 2012 11785ndash92

Lot CountrySource Genotype

1437Z

vial

USA Connecticut HPV-11

HPV-18

1511Z prefilled syringe

USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18

NL35360 prefilled syringe

France HPV-11

HPV-18

NP23400 prefilled syringe

Spain Valencia

HPV-11

HPV-18

NN33070 prefilled syringe

Spain Valencia

HPV-11

HPV-18

NM25110 prefilled syringe

Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe

New Zealand Tauranga HPV-18

NK16180 prefilled syringe

New Zealand Northland HPV-18



HPV-18


New Zealand

Christchurch

HPV-11

HPV-18


New Zealand Wellington HPV-11

HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8

Five Gardasil vaccine samples from New Zealand physicians

-all tested positive for HPV L1 gene DNA

(HPV-16 DNA in non-B conformations)

9

Degenerate (MY11) and consensus (GP6) PCR primer amplification of HPV L1 DNA

10

HPV

Type

ID Locus

Nucleotide Sequence (3rsquo ndash 5rsquo) Total

bases

Location

(5rsquo ndash 3rsquo) GP6 site MY11 site

6 AF092932 GAAAAATAAATTGTAAATCA ----- CCATTGTTATGTCCCTGGGC 181 6723 - 6903

11 EU918768 GAAAAATAAACTGTAAATCA ----- CCATTGTTATGTCCCTGAGC 181 6707 - 6887

16 FJ006723 GAAAAATAAACTGTAAATCA ----- CCATTATTGTGGCCCTGTGC 184 6641 - 6824

18 GQ180792 GAAAAATAAACTGCAAATCA ----- CCATTGTTATGACCCTGTGC 187 6558 - 6744

26 NC_001583 GAAATATAAATTGTAATTCA ----- CCATTATTATGACCCTGTGC 187 6533 - 6719

31 PPH31A GAAATATAAATTGTAAATCA ----- CCATTATTGTGTCCCTGAGC 184 6500 - 6683

32 X74475 GAAATATAAACTGTATATCA ----- CCATTATTGTGGCCTTGTGC 181 6817 - 6997

33 EU 918766 GAAAAACAAACTGTAGATCA ----- CCATTATTATGACCTTGTGC 181 6542 - 6722

35 PPH35CG GAAAAATAAACTGTAAATCA ----- CCATTATTATGGCCTTGTGC 184 6522 - 6705

39 PPHT39 GAAATATAAATTGTAAATCA ----- CCATTGTTGTGGCCCTGGGC 187 6585 - 6771

40 X74478 GAAAAATAAACTGCAAATCA ----- CCATTGTTATGGCCCTGGGC 187 6718 - 6904

45 EF202167 GAAAAATAAACTGTAAATCA ----- CCATTGTTATGGCCCTGGGC 187 6562 - 6748

51 PPHDNA GAAAAATAAATTGCAATTCA ----- CCATTATTGTGACCCTGCGC 184 6466 - 6649

52 GQ472848 GAAAAATAAATTGTAAATCG ----- CCATTATTGTGGCCCTGCGC 181 6631 - 6811

53 NC 001593 GAAACACAAATTGTAATTCA ----- CCATTATTATGTCCCTGGGC 181 6594 - 6774

54 AF436129 GAAATATAAATTGTAAATCA ----- CCATTGTTCTGACCCTGGGC 181 6509 - 6689

55 HPU31791 GAAACATAAACTGTAAGTCA ----- CCATTATTGTGGCCCTGCGC 187 6625 - 6813

56 X74483 GAAAAACAAATTGTAATTCA ----- CCATTATTATGGCCTTGGGC 181 6539 - 6719

58 FJ407217 GAAAAACAAACTGTAAGTCA ----- CCATTGTTATGACCTTGTGC 181 6588 - 6768

59 EU918767 GAAATATAAACTGCAAATCA ----- CCATTGTTTAAACCCTGAGC 187 6551 - 6737

61 HPU31793 GAAAAATAAATTGCAAATCA ----- CCATTGTTGTGGCCCTGGGC 184 6712 - 6895

62 AY395706 GAAATATAAATTGCAAATCA ----- CCATTATTATGGCCCTGCGC 181 6725 - 6905

66 EF177191 GAAACACAAACTGTAGTTCA ----- CCATTATTATGGCCCTGTGC 181 6590 - 6770

67 D21208 GAAATATAAACTGCAAATCA ----- CCATTGTTATGACCTTGTGC 181 6564 - 6744

68 EU918769 GAAATATAAATTGCAAATCA ----- CCATTGTTGTGTCCCTGTGC 187 6453 - 6639

69 AB027020 GAAATATAAACTGTAATTCA ----- CCATTATTATGACCCTGGGC 187 6489 - 6675

70 HPU21941 GAAATATAAATTGTAAATCA ----- CCATTATTGTGTCCCTGGGC 187 6529 - 6715

71 AY330623 GAAATATAAATTGCAAATCA ----- CCATTGTTTGTGCCCTGTGC 181 6791 - 6971

72 X94164 GAAATATAAACTGCAAATCA ----- CCATTGTTGTGACCCTGGGC 184 6738 - 6921

73 X94165 GAAAAACAAACTGTAAATCA ----- CCATTATTTTGTCCCTGTGC 190 6433 - 6622

74 AF436130 GAAAAATAAATTGCAAATCA ----- CCATTATTGTGGCCTTGCGC 190 6524 - 6713

81 AJ620209 GGAAAATAAACTGCAAATCA ----- CCATTATTATGGCCCTGTGC 181 6809 - 6989

83 AF151983 GCAATATAACCTGTAAGTCA ----- CCATTATTATGTCCCTGGGC 180 6693 - 6872

84 AF293960 GGAATATAAACTGCAAATCA ----- CCATTGTTATGACCCTGGGC 181 6619 - 6799

86 AF349909 GAAAAATAAATTGCAAATCA ----- CTATTATTGTGTCCCTGCGC 181 6615 - 6795

87 AJ400628 GAAAAATAAACTGTAAATCA ----- CCATTATTGTGGCCCTGGGC 181 6712 - 6892

89 AF436128 GGAATATAAACTGTAGGTCA ----- CCATTATTATGGCCCTGTGC 180 6706 - 6885

91 AF419318 GAAATATAAACTGTAAATCA ----- CCGTTATTATGCCCCTGTGC 187 6810 - 6996

DNA sequencing of mixed HPV-16 and HPV-18 L1 genes in

B conformation

The size of the GP6MY11 PCR amplicon of HPV-18 is 3-bp longer than that of

HPV16 Therefore one end of the primer sequence 3rsquoCCATT---5rsquo of the MY11

primer site is positioned ahead of the other as shown in this mixed HPV DNA

sequence tracing The first T of each end segment is indicated by an arrow

Lee SH Methods Mol Biol 2012 90365-101

11

12

Specific DNA sequence of HPV-16 L1 gene DNA in non-B conformation mixed with

HPV-18 L1 DNA in Gardasil lot NL01490 was amplified with a modified MY11+ primer

HPV-16 L1 gene DNA in non-B conformation found in postmortem

blood and spleen of an 18 years old female who died in sleep 6

months after Gardasil vaccination in New Zealand

Amorphous aluminum hydroxyphosphate

sulfate adjuvant and phosphate ligand

exchanges Egan et al on Hep B vaccinesVaccine 27 (2009) 3175ndash3180

Schematic representation of the E2 DNA binding domain from BPV-1 bound to DNA HPV topological non-B Conformation in nature

Lima L M T R and de Prat-Gay G J Conformational Changes and Stabilization Induced by Ligand Binding in the DNA-binding Domain of the E2 Protein from Bovine PapillomavirusBiol Chem 199727219295-19303

copy1997 by American Society for Biochemistry and Molecular Biology

HPV VLPs adsorbed to AAHS nanoparticles httpwwwnanoimagingservicescomCaseStudiesCharacterization‐of‐Gardasilaspx

17

Calcium Phosphate and Aluminum Hydroxide as Non-Virus Gene

Carrier The Morphology of DNA-salt Complex and the Effects It on

DNA Transfection KAGAKU KOGAKU RONBUNSHU 2003 29680-684

We also investigated the complex between DNA and aluminum

hydroxide which already used as an adjuvant The extent of DNA

uptake was also affected by the size of the DNA-aluminum hydroxide

complex The dramatic changes in DNA conformation are discussed in

relation to the efficiency of gene transfection

Transfection process of deliberately introducing nucleic acids into

eukaryotic cells by non-viral methods

Double Edged Sword of DNAAluminum Complexes

bull Host DNAaluminum adjuvant complex phagocytized by APCs

activates pathways that increase their ability to interact productively

with antigen-specific CD4 T cells [12] ^ host immune responses

bull What does viral DNAaluminum complex do

(a) Free HPV-16 L1 gene DNA injected into mice induces a strong CD8 T cell

response [3]

(b) Viral DNA activates the macrophages through the interferon regulatory

transcription factor IRF3 (TLR) to release cytokines including TNF

(c) TNF is a myocardial depressant and promotes inflammation

Macrophages know host DNA harmless viral DNA harmful thus react differently

References

[1] Marichal T et al Nat Med 2011 17 996-1002

[2] McKee AS et al Proc Natl Acad Sci USA 2013 110 E1122-E1131

[3] Caulfield MJ et al Human Vaccines 2007 3 139-145

18

Viral DNA Toll-like Receptor (IRF3-dependent) activation Goutagny N Severa M Fitzgerald KA Nat Immunol 2006 Jun7(6)555-7

19

20

J Casanova L Abel The human model a genetic dissection of immunity to infection in natural

conditions Nature Reviews Immunology 2004 4 55-66

Crosstalk between Complement and Toll-Like Receptors

(alternative pathwayviralbacterial DNA properdin Crsquo3) initiated by pathogen-associated molecular patterns

W-C SONG Toxicol Pathol 2012 40 174

What can a mother expect

bull Some neurological damages such as loss of vision and other

functions due to neuronal damages are not reversible

bull Cardiac functions probably return to normal when the HPV DNA

is finally degraded Why is there such a hope

[1] The HPV-16 antibody titers stay high for 5 years after vaccination

but the HPV-18 titers drop such that one-third of women lose their

seropositivity 2 years suggesting that the viral DNAaluminum adjuvant

degraded during this period

[2] All reported unexpected sudden deaths after Gardasil vaccination

seemed to occur in women before age 20

What can a mother do

bull Find a sympathetic doctor who is willing to

listen Educate your doctor if possible- it is

not psychosomatic

bull Realize that your doctor may not be in a

position to help due to political pressures

So you have to be sympathetic too

24

Professional risk for

openly challenging the

status quo of a

dysfunctional system

- a personal experience

SANE Vax Inc and Sin Hang

Lee MD Offers American

Consumers Reliable HPV

Genotyping - NCI Pays a Foreign

Laboratory for Similar Testing

httpwwwbusinesswirecomne

wshome20100920005586enSA

NE-Vax-Sin-Hang-Lee-MD-

Offers

Lee SH From human

papillomavirus to cervical

cancer Obstet Gynecol 2010

Nov116(5)1221 author reply

221-2

25

Properdin-A Short Review Sin Hang Lee Chinese Medical Journal

Received 9-24-1957 Published 1958

Louis Pillemer-humiliated ruled suicide 8-31-1957

Freedom Through Truth For Service School motto ndash former Yenching University (Beijing)

John Leighton Stuart

bull Educator diplomat missionary

bull Born in China 1876

bull Died in America 1962

bull Ashes eventually buried in

China -2008 as he wished

27

Pathology is a specialty of Medicine

bull Why or how people die

bull Why or how people get sick

bull Analyze patient samples for diagnosis

bull Molecular pathology

Sanger sequencing DNA analysis for

diagnosis of HPV Chlamydia gonorrhea

Lyme disease Ebola and breast cancer gene

mutations ndash

Precision Molecular Personalized Medicine

2



4
































Court MI

5





Neurol 2010 25321-327









7




1437Z

vial


HPV-18


USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18


France HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe






HPV-18


New Zealand

Christchurch

HPV-11

HPV-18



HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27



4
































Court MI

5





Neurol 2010 25321-327









7




1437Z

vial


HPV-18


USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18


France HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe






HPV-18


New Zealand

Christchurch

HPV-11

HPV-18



HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27











Court MI

5





Neurol 2010 25321-327









7




1437Z

vial


HPV-18


USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18


France HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe






HPV-18


New Zealand

Christchurch

HPV-11

HPV-18



HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27





Neurol 2010 25321-327









7




1437Z

vial


HPV-18


USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18


France HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe






HPV-18


New Zealand

Christchurch

HPV-11

HPV-18



HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27




1437Z

vial


HPV-18


USA New York

HPV-18

0553AA

vial

USA New Jersey

HPV-11

HPV-18


France HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Spain Valencia

HPV-11

HPV-18


Australia Sydney

HPV-11

HPV-18

NL01490 prefilled

syringe






HPV-18


New Zealand

Christchurch

HPV-11

HPV-18



HPV-18


Poland

HPV-11

NN28160

vial

Russia

HPV-11

HPV-18


Bulgaria

HPV-11

HPV-18


India HPV-18

8




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27




9


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27


10

HPV

Type

ID Locus


bases

Location









































B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27


B conformation






11

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27

12













17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27











17

















response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27







response [3]





References




18


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27


19

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27

20




















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27


















not psychosomatic




24



status quo of a











Offers

Lee SH From human




221-2

25











27



status quo of a











Offers

Lee SH From human




221-2

25











27











27

viral dna bound to aluminum may mediate hpv vaccination ...8 . five gardasil vaccine samples from...

Documents