A same-nested PCR was used to re-amplify the amplicon of a hypervariable region of the HPV-16 L1 gene DNA in the postmortem blood and splenic tissue obtained at autopsy of a formerly healthy teenage girl who suffered a sudden unexpected death in sleep 6 months after 3 intramuscular injections of a quadrivalent HPV vaccine, Gardasil ?. A full autopsy analysis revealed no cause of death. The HPV-16 gene DNA detected in the postmortem materials was similar to the HPV-16 gene DNA fragments in Gardasil ? in that both were in non-B-conformation, requiring nondegenerate GP6 and MY11 primers to re-amplify the PCR amplicon for detection and to generate a template useful for direct DNA sequencing. A sequence excised from the base-calling DNA sequencing electropherogram was analyzed by Basic Local Alignment Search Tool (BLAST) alignment and a 45 - 60 base sequence fully matched with a standard hypervariable region of the HPV-16 L1 gene retrieved from the National Center for Biotechnology Information database validated the correct genotyping for HPV- 16 L1 gene DNA. These naked non-proliferating HPV- 16 L1 gene DNA fragments appeared to be in the macrophages of the postmortem blood and spleen, and were protected from degradation by binding firmly to the particulate aluminum adjuvant used in vaccine formulation. The significance of these HPV DNA fragments of a vaccine origin found in post-mortem materials is not clear and warrants further investigation.
Virus-like particles (VLPs) of human papillomavirus (HPV) are irregularly shaped 30 - 50 nm structures composed of self-assembled HPV major capsid L1 protein pentamers manufactured by a DNA recombinant technology [1,2]. Genotype-specific VLPs for HPV-16, -18, -11 and -6 are used as the active ingredient of a quadrivalent HPV vaccine Gardasil® which has been shown to reduce the incidence of cervical intraepithelial neoplasia grade 2 and grade 3 in vaccinated women [
On the other hand, the estimated rate of anaphylaxis in the young women receiving Gardasil® vaccination is also high and has been reported to be 5 to 20 times those identified in comparable school-based vaccination programs, using the Brighton case definition of anaphylaxis for diagnostic certainty [
The parents of a formerly healthy New Zealand young woman who suffered a sudden unexpected death in sleep 6 months after Gardasil® vaccination requested testing for the presence of HPV L1 gene DNA in the post-mortem samples of their deceased daughter collected at the time of autopsy. Some of the consultants to the parents suggested that if residual HPV L1 gene DNA which is known to be present in the Gardasil® vaccine [8,9] were present in the postmortem samples, there might be a potential link between the residual HPV DNA and the unexplained death of their daughter. This paper reports the experience in developing a method for the detection and validation of minute quantities of HPV-16 L1 gene DNA in the postmortem blood and spleen obtained at autopsy. The data reported in this paper were extracted from a full report which was submitted to the Wellington coronial court at a public inquest held on August 8-9, 2012.
The parents of the deceased have granted their permission to the author to publish the data contained in this report.
An 18-year-old healthy woman living with her parents was found dead in bed. The only relevant medications that she received before death were three injections of Depo Provera over a period of four years and three doses of intramuscular injections of the HPV vaccine, Gardasil®, in the last year of her life with the last dose of Gardasil® vaccination given six months prior to her demise. There was no history of alcohol or drug abuse. According to the documents presented at the inquest, the patient experienced temperament changes shortly after the first dose of Gardasil® injection, started to have dizziness spells, pins-and-needles feelings in her hands, memory lapses and abdominal pains after the second injection, and developed intermittent weak arm, frequent tiredness requiring daytime naps, increased pins-and-needles feelings in hands causing things to drop from hands, appetite increase with no weight gain, night sweats, loss of ability to use common objects, intermittent chest pain and sudden unexpected “racing heart”. A full autopsy analysis revealed no anatomical, histological, toxicological, genetic or microbiological findings that might be linked to a potential cause of death.
At the parents’ request and by order of the coronial court, DNA samples of the postmortem blood and splenic tissue of the deceased were prepared by Dr. Donald Love at Auckland Hospital Molecular Genetics Laboratory to be tested for the presence of HPV L1 gene DNA. According to Dr. Love, the commercial Gentra® Puregene® Blood Kit (Qiagen) was used to extract the DNA from the nucleated cell fraction of the unfixed blood and splenic tissue which were obtained at the time of autopsy and had been stored at –80˚C. The purified DNA was finally dissolved in TE buffer at the concentration of 0.5 µg of DNA per µL, speedvac dried in plastic tubes, and sent to the author’s laboratory for analyses. Based on the second edition of Gentra® Puregene® Handbook (September 2007), the DNA yield with this method of preparation is about 6 pg DNA per human diploid cell. Therefore, 0.5 µg of DNA was equivalent to the amount of DNA extracted from ~80,000 nucleated cells (500 ng/6pg). A few split samples of the dried DNA are stored at Auckland Hospital for possible future investigation by independent laboratories at the order of the coronial court.
The traditional heat-resistant Taq DNA polymerase could not generate a useful nested PCR amplicon from a minute quantity of target HPV DNA in the postmortem materials to be used as a template for direct DNA sequencing. As a result, a LoTemp® PCR with a highly processive HiFi® DNA polymerase system programmed at thermocycling steps not to exceed 85˚C was selected for this study. The general method used to detect HPV L1 gene DNA by heminested (nested) LoTemp® PCR amplification with the GP/MY degenerate consensus primers and validation with direct automated DNA sequenceing for genotyping has been described in detail elsewhere for clinical samples [20-25] and for detecting residual HPV DNA fragments in the Gardasil® vaccine [
Transferring of PCR products was accomplished by a micro-glass rod to eliminate the need for micropipetteting to avoid aerosol contamination [
A “same-nested” PCR was introduced for re-amplification of a target region of an HPV L1 gene in the postmortem samples in this case. To perform a same-nested PCR, the primary PCR and the subsequent same-nested PCR(s) were conducted with an identical pair of PCR primers, or the subsequent same-nested PCR was conducted with a pair of the same primers having a few new bases added to the 3’end for one or for both of the primers which had been used in the prior PCR. As a result, all same-nested PCR products were terminated by the first pair of PCR primers used to initiate the primary PCR. The same-nested PCR protocol was found to be necessary to amplify the HPV-16 L1 gene DNA fragments in the postmortem materials in this case and the HPV-16 L1 gene DNA fragments in the Gardasil® vaccine [
After completion of the primary and the nested PCR, a 5 µL aliquot of the PCR products was pipetted out from each tube and mixed with 2 µL loading fluid for electrophoresis in a 2% agarose gel containing ethidium bromide. The gel was examined under UV light for various PCR product bands in the agarose gel.
For DNA sequencing, a trace of the positive nested PCR product was transferred directly with a micro-glass rod from the positive nested PCR tube into a 20 µL volume of a cycle sequencing reaction mixture consisting of 14.5 µL water, 3.5 µL of 5× buffer, 1 µL of BigDye Terminator 1.1 (Applied Biosystems) and 1 µL of 10 µM sequencing primer. After thermal cycling according to the manufacturer’s recommendation, the reaction mixture was loaded in an automated ABI 3130 four-capillary Genetic Analyzer for sequence analysis. Alignment analysis of a 45 - 60 base sequence in the hypervariable region of the L1 gene excised from the computer-generated base-calling electropherogram was performed against various standard HPV genotype sequences stored in the GenBank, using the on-line BLAST (Basic Local Alignment Search Tool) system to validate the specific HPV genotyping.
The PCR and DNA sequencing primers used and referred to in this report with their DNA sequences are listed as follows.
Degenerate HPV L1 gene primers:
MY09 = 5’-CGTCCMARRGGAWACTGATC-3’
MY11 = 5’-GCMCAGGGWCATAAYAATGG-3’
Key to degenerate nucleotides: M = (A + C), R = (A + G), W = (A + T), Y = (C + T)
Non-degenerate HPV L1 gene primers:
GP5 = 5’-TTTGTTACTGTGGTAGATAC-3’
GP6 = 5’-GAAAAATAAACTGTAAATCA-3’
GP6+ = 5’-GAAAAATAAACTGTAAATCATATTC- 3’
MY11 (1) = 5’-GCACAGGGACATAACAATGG-3’
MY11 (2) = 5’-GCACAGGGACATAATAATGG-3’
MY11 (3) = 5’-GCACAGGGTCATAACAATGG-3’
MY11 (4) = 5’-GCACAGGGTCATAATAATGG-3’
MY11 (5) = 5’-GCCCAGGGACATAACAATGG-3’
MY11 (6) = 5’-GCCCAGGGACATAATAATGG-3’
MY11 (7) = 5’-GCCCAGGGTCATAACAATGG-3’
MY11 (8) = 5’-GCCCAGGGTCATAATAATGG-3’
HPV16MY11+ = 5’-GCACAGGGCCACAATAATGGCAT-3’
The choice of an appropriate combination of PCR primers at different stages was crucial to generate a relatively pure template useful for direct DNA sequencing. Lengthening a PCR primer increased the specificity of target DNA amplification at the expense of sensitivity in this case. The purified full-length HPV-16 plasmid DNA purchased from American Type Culture Collection, diluted to a concentration of 1 copy of HPV-16 L1 gene DNA per µL in TE buffer, was used as the positive control. At the latter theoretical concentration, about 50% of the primary PCRs and 100% of the same-nested PCRs would generate a PCR amplicon of ~190 bp in size at agarose gel electrophoresis when 1 µL of the HPV-16 positive control was used as the template and the degenerate consensus 20-base GP6/MY11 primer pair as the same-nested PCR primers.
To detect minute quantities of HPV L1 gene DNA fragments in the whole blood DNA, 1 µL of undiluted reconstituted sample containing 0.5 µg of human genomic DNA was used to start each primary PCR. The general protocol of MY09/MY11 degenerate primer amplification followed by degenerate consensus GP6/MY11 heminested PCR amplification, which has proved highly successful in detecting HPV DNA and in preparing templates for direct DNA sequencing for testing HPV DNA in clinical specimens [
To generate an amplicon suitable for DNA sequencing validation, a second same-nested PCR was performed, using a pair of HPV16MY11+/GP6 primers for re-amplification (
In order to characterize the non-target PCR products resulting from a GP6/MY11 primer nested PCR amplification, the amplicon illustrated in lane 2 of
Based on the above experiments, a same-nested PCR
with a pair of 20-base non-degenerate MY11 (1)/GP6 primers was chosen for the initial detection of HPV-16 L1 gene DNA fragments in all batches of DNA extraction on this case. It turned out that only about 1/3 of the same-nested PCRs with this protocol showed a positive amplicon of HPV-16 L1 gene DNA, eventually validated by DNA sequencing. The non-target human genomic DNA fragments which were co-amplified in the samenested PCR varied considerably. For example, in a splenic DNA extract, when four 1 µL aliquots of the reconstituted DNA sample were used for a parallel same-nested PCR experiment, all using the non-degenerate MY11 (1)/GP6 primer pair for amplification, and two of the PCRs were cycled under a low stringency condition with a 40˚C annealing temperature while two under a high stringency condition with a 50˚C annealing temperatureonly one of the PCRs generated a ~190 bp HPV DNA amplicon. Even in the latter nested PCR, there was a heavy ~500 bp product which was the result of co-amplification of non-target DNA in a heminested PCR setting (
A same-nested PCR in which one identical pair of nondegenerate primers selected from the well-characterized degenerate consensus 20-base GP6/MY11 primer group, referred to as the MY11 (1)/GP6 primers in this report, was needed to detect the HPV-16 L1 gene DNA fragments present in the postmortem blood and the splenic tissue obtained at autopsy of a teenage girl who suffered a sudden unexpected death in sleep 6 months after Gardasil® vaccination. Since the human genomic samples contain numerous DNA fragments which are substantially complementary to the base sequences of the HPV PCR primers, co-amplification of non-target DNAs of the human genome invariably occurs in the same-nested PCR settings when PCR amplicons are re-amplified with the same primer(s). These human genomic DNA segments may act as primer-binding PCR inhibitors even when the partially matched primer-binding does not generate PCR amplicon bands visible at agarose gel electrophoresis. The same-nested PCR procedure has the effects of reducing the concentration of inhibitors carried over from the original sample by simple dilution so that the chance of obtaining a target DNA amplicon is significantly increased [
The elongated HPV16MY11+/HPV16GP6+ primers cannot be used to generate a PCR amplicon directly from the HPV-16 DNA template in the postmortem materials in this case. In comparison, the 184-bp L1 gene DNA
template in the HPV-16 plasmid DNA control and in the HPV-16 DNA isolated from clinical cervicovaginal cytology samples is always successfully amplified by the 20-base degenerate consensus GP6/MY11 primer pair and by the elongated HPV16MY11+ /HPV16GP6+ primer pair under identical same-nested PCR conditions. Unlike the L1 gene in the HPV-16 plasmid DNA and in the HPV-16 isolates from clinical cervicovaginal samples, the HPV-16 L1 gene DNA fragments found in the postmortem blood and splenic samples cannot be amplified under low stringency PCR condition and lacks a useful MY09 primer-binding site for PCR amplification. These variances in PCR amplification characteristics indicate that there are topological conformational changes in the HPV-16 L1 gene DNA fragments in the postmortem samples. Similar topological non-B conformational changes in HPV L1 gene DNA fragments bound to the AAHS particles in the Gardasil® vaccine have been demonstrated by a low temperature (LoTemp®) PCR catalyzed by a highly processive DNA polymerase with proof-reading function [
DNA molecule [
Gardasil® is a quadrivalent vaccine which is known to contain residual recombinant HPV L1 gene DNA fragments [
HPV-16 is a virus which only infects human mucosal epithelial cells. HPV-16 DNA may be detected in the plasma of patients with invasive squamous cervical cancer harboring the same genotype of virus, but not in the control subjects without cervical cancer [
The HPV-16 L1 gene DNA fragments detected in the postmortem blood and splenic tissue in this case are presumably in minute quantities and in the nucleated cells, probably macrophages. Naked viral and bacterial DNA fragments firmly bound to insoluble aluminum salts can be carried into tissue macrophages through phagocytosis to initiate a series of DNA-related immune reactions [31-34]. Intramuscular injection of free HPV-16 L1 plasmid DNA in BALB/C mice without adjuvant has been known to induce a strong CD8 T cell response [
The presence of HPV-16 L1 gene DNA fragments of a vaccine origin indicates possible co-existence of other companion microbial DNA, such as DNA fragments of the plasmid pGAL110 and yeast cells which are used in the vaccine production by the manufacturer [
phages [41-46] is to cause release of various cytokines, including tumor necrosis factor (TNF), a recognized myocardial depressant [47-51]. TNF-induced hypotensive shock is a documented observation among animals
[52,53] and humans [54,55]. To answer the question whether the quantity of these persistent viral or microbial DNA fragments can stimulate the macrophages to release enough TNF to generate a significant pathophysiological impact following Gardasil® vaccination needs expanded research.
Detection of HPV-16 L1 gene DNA fragments in non-Bconformation in postmortem blood and spleen from a person who died suddenly and unexpectedly 6 months after quadrivalent HPV vaccination has not been previously reported and warrants further investigation.
This study was commissioned and sponsored by SANE VAX, Inc. for a future payment not to exceed one US dollar. The author thanks Ms. Veronica S. Vigliotti and Ms. Jessica S. Vigliotti for donating their extremely valuable technical and professional time to assist completion of this study.