A low temperature (LoTemp?) polymerase chain reaction (PCR), conducted at cycling temperatures not to exceed 85℃and catalyzed by a novel highly processive HiFi? DNA polymerase with proofreading function, was used to study the topological conformational changes of the human papillomavirus (HPV) L1 gene DNA fragments bound to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant in the quadrivalent HPV vaccine, Gardasil?. L1 gene DNA fragments of HPV-11, HPV-18 and HPV-16 were detected in the AAHS particles by nested PCR, but all were lacking a region that was amplifiable by an MY09 degenerate primer. In addition, a pair of degenerate consensus GP6/MY11 primers was able to amplify a target segment of the HPV-11 L1 gene DNA and the HPV-18 L1 gene DNA bound to the AAHS particles as expected for any HPV DNA in the B-conformation. However, there was no co-amplification of the HPV-16 L1 gene DNA known to coexist in the same samples. The lack of co-amplification was verified by direct DNA sequencing of the PCR amplicons. The companion HPV-16 L1 gene DNA in the same sample required repeated PCRs with a pair of modified non-degenerate GP6/ MY11 primers for detection. This melting profile of the HPV-16 L1 gene DNA was similar to that of the HPV-16 L1 gene DNA recently discovered in the postmortem blood of a young woman who suffered a sudden unexpected death 6 months after Gardasil? vaccination. The findings suggest that the topological conformational changes in the HPV L1 gene DNA residues bound to the AAHS adjuvant may be genotype-related. The special non-B-conformation may prevent the HPV-16 L1 gene DNA from being degraded in the body of the vaccine recipients after in- tramuscular injection.
Molecular characterization of human papillomavirus (HPV) DNA in clinical specimens begins with primerdirected enzymatic polymerase chain reaction (PCR) amplification of the target DNA with a thermostable DNA polymerase. In practice, pre-PCR purification of DNA samples is required [1-4], and the DNA template must be soluble in an aqueous solution. When the viral load is low, attempts to purify a minute quantity of DNA from a complex sample may risk losing the target template all together.
A low temperature (LoTemp®) PCR system with a highly processive DNA polymerase which also has proof-reading function has been used to increase the sensitivity of PCR detection of HPV DNA in proteinase-K digested unpurified clinical samples without sacrificing specificity [5,6]. This novel PCR system depends on chemical denaturation of the dsDNA template at 85˚C so that the highly stable but less heat-resistant HiFi® DNA polymerase of high processivity can perform its function of cyclic primer extension at 65˚C. In this system, the annealing temperature is set at 50˚C for high stringency PCR, and at 40˚C for low stringency PCR amplification. Using LoTemp® PCR, residual HPV L1 gene DNA fragments have been detected in the proteinase K-digested insoluble fraction of the HPV vaccine Gardasil® (Merck and Co., Inc.) [
A total of 16 Gardasil® vials or manufacturer-prefilled vaccine syringes with intact original packages were purchased by licensed practitioners in nine countries and submitted to the author’s laboratory to be tested for residual HPV L1 gene DNA fragments. The first part of the test results based on degenerate consensus primer nested PCR amplification was previously reported [
An aliquot of 100 μL of the vaccine suspension was centrifuged at ~16,000 × g for 10 min in a 1.5 mL microcentrifuge tube. The pellet was washed twice with 1 mL of 70% ethanol each and the final ethanol suspension was centrifuged at ~16,000 × g for 5 min. The washed pellet was air-dried. The dried pellet was re-suspended in 100 μL of 0.1 mg/mL proteinase K (Sigma Chemical Co., St. Louis, MO) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, 0.5% Tween 20, pH 8.1. The mixture was digested at 45˚C - 55˚C overnight and was exhaustively washed with the same Tween 20 buffer pH 8.1, 4 times, 1 mL each time. At this pH, both the AAHS particles and the DNA molecules carry a negative electrostatic charge. The final washed pellet, presumably consisting of protein-free AAHS particles, was resuspended in 100 μL of buffer. After heating at 95˚C for 10 min, 1 μL of the washed and heated insoluble particle suspension was used for each primary PCR followed by nested PCR.
Initially, a Taq DNA polymerase was used for the PCR amplification. In this Taq PCR protocol, 1 mL of the sample suspension, 2.5 units of GE rTaq DNA polymerase in 0.25 mL (GE Healthcare, Piscataway, NJ, USA), 2.5 mL of 10× PCR buffer, 2 mL of 25 mM MgCl2, 1 mL of 5 mM dNTPs, 1 mL of MY09 primer (10 mM), 1 mL of MY11 primer (10 mM), and water were added up to a final volume of 25 µL to initiate a primary PCR. The nested PCR mixture contained the same ingredients except that the primers were GP6/MY11 or GP5/MY09 and the 1 mL of the suspension was replaced with water. The primary PCR products were transferred to the nested PCR tubes by micro-glass rods to avoid micropipetting aerosol [
For the low temperature PCR, 20 µL of the LoTemp® master mix containing manufacturer-optimized HiFi® DNA polymerase, magnesium ions, denaturing agents, and dNTPs with stabilizing additives was used to replace the GE rTaq DNA polymerase, the buffer, the MgCl2 solution, and dNTPs described for the Taq PCR in a final volume of 25 µL. The amounts of template materials and the primers were identical to those described for the Taq PCR protocol. The LoTemp® thermocycling steps were set for an initial heating at 85˚C for 10 min, followed by 30 cycles, each set at 85˚C for 30 sec, 40˚C for 30 sec, and 65˚C for 1 min. The final extension was 65˚C for 10 min.
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 basecalling 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 and for visual sequence analyses.
The sequences of the well characterized degenerate [
MY09 = 5’-CGTCCMARRGGAWACTGATC-3’
MY11 = 5’-GCMCAGGGWCATAAYAATGG-3’
Key to degenerate nucleotides: M = (A + C), R = (A + G), W = (A + T), Y = (C + T)
GP5 = 5’-TTTGTTACTGTGGTAGATAC-3’
GP6 = 5’-GAAAAATAAACTGTAAATCA-3’
GP6+ = 5’-GAAAAATAAACTGTAAATCATATTC- 3’
MY11(1) = 5’-GCACAGGGACATAACAATGG-3’
HPV16MY11+ = 5’-GCACAGGGCCACAATAATGGCAT-3’
The binding sites for the four principal primers in the HPV L1 gene ORF are positioned in following order:
5’---MY11----GP5-----GP6----MY09---3’
In this report, nested PCR refers to heminested PCR with the GP6/MY11 or the GP5/MY09 primer pair, except the “same-nested PCR” defined below.
A “same-nested” PCR was designed for amplification of a 184 bp hypervariable region of an HPV-16 L1 gene DNA in the vaccine and in the postmortem blood of a young woman who suffered a sudden unexpected death 6 months after Gardasil® vaccination. For the same-nested PCR protocol, the primary PCR and the subsequent same-nested PCR(s) were conducted with an identical pair of non-degenerate 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 of one or to 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 postmortem blood DNA was prepared by Dr. Donald Love at Auckland Hospital Molecular Genetics Laboratory in New Zealand and sent to the author’s laboratory for HPV DNA testing by the order of the Wellington coronial court in connection of a public inquest hearing on August 8-9, 2012.
Both conventional Taq polymerase PCR and the low temperature PCR with HiFi® DNA polymerase amplified the control HPV-16 plasmid DNA to generate an amplicon of ~450 bp in the primary PCR with the degenerate MY09/MY11 primers and an amplicon of ~190 bp in the nested PCR with the degenerate consensus GP6/MY11 primers, respectively (
When the degenerate MY09/MY11 primary PCR products were blindly transferred to the PCR mixture containing the GP5/MY09 primer pair for nested PCR amplification, no visible nested PCR product band was obtained at agarose gel electrophoresis (
The HPV-16 L1 gene DNA bound to AAHS particles
appeared to acquire a non-B-conformation different from that of the HPV-11 and HPV-18 DNA. This phenomenon was further illustrated by PCR and DNA sequencing on Gardasil® vaccine lot #NL01490 which proved to contain both HPV-18 L1 gene DNA fragments and HPV-16 L1 gene DNA fragments. The degenerate consensus GP6/MY11 primer nested PCR product of Gardasil® lot #NL01490 was illustrated in lane 1, HiFi nest,
There was no evidence of co-amplification of any other HPV DNA by the 20-base degenerate MY11 primer, which was underlined on the right end with the three degenerate bases marked by a yellow dot on the top of each degenerate base.
In the course of this study, Gardasil® lot sample #NL01490 was found to also contain HPV-16 L1 gene DNA fragments which required two same-nested PCRs to amplify, namely one primary PCR and one samenested PCR with a pair of non-degenerate HPV16- MY11+/GP6 primers and a second nested PCR with a pair of non-degenerate HPV16MY11+/GP6+ primers to obtain a template (
Numerous attempts to amplify an HPV L1 gene DNA in the postmortem blood with a pair of degenerate consensus GP6/MY11 primers under high stringency and low stringency PCR condition failed. After several experiments, 0.5 µg of the extracted genomic DNA was chosen to start a same-nested PCR under high stringency
condition with a pair of non-degenerate MY11(1) and GP6 primers. Only with a second same-nested PCR when the elongated 23-base non-degenerate HPV16- MY11+ was paired with a GP6 primer, a faint HPV DNA amplicon of ~190 bp was generated in 3 of 10 samples (
Residual HPV L1 gene DNA in the vaccine Gardasil® [
The DNA sequences of the type-specific HPV L1 genes used to manufacture the Gardasil® vaccine are well known. The highly conserved region of the HPV L1 gene, about 450 bp in size and terminated by the binding sites for the degenerate MY09 and MY11 primers, is well characterized. Heminested PCR based on degenerate consensus GP6/MY11 and GP5/MY09 amplification has been used for routine detection and genotyping of various HPV genotypes in clinical specimens [5,6,23, 24]. The hypervariable 181 - 187 bp L1 gene segment of HPV-11, HPV-16 and HPV-18 in the B-conformation is invariably amplified by the degenerate consensus GP6/ MY11 primer pair individually or co-amplified if more than one HPV genotype is present in a sample [6,23,24]. The lack of GP5/MY09 PCR primer amplification (
The degenerate consensus MY09/MY11, GP5/MY09 and GP6/MY11 PCR primer pairs all fail to amplify the HPV-16 L1 gene DNA fragments present in the Gardasil® vaccine lot samples tested [Manuscript under peer review]. In the current study, experiments were designed to use the degenerate consensus GP6/MY11 primer pair
to amplify the HPV DNA in a vaccine sample known to contain both HPV-18 L1 gene DNA and HPV-16 L1 gene DNA in order to confirm that variable topological conformational changes in DNA of different genotypes may take place when bound to an insoluble aluminum salt. The results showed that only an HPV-18 amplicon was generated (
The presence of HPV DNA fragments of vaccine origin in the body of vaccine recipients might be anticipated after intramuscular injections of Gardasil® since the vaccine is known to contain these DNA fragments [
This is the first time that PCR amplification followed by direct DNA sequencing has been used to study topological conformational changes in DNA fragments
bound to an insoluble aluminum salt. The study has been conducted with a low temperature PCR system in nested PCR settings. It may require more than one nested PCR to generate a useful template for direct DNA sequencing analysis when the initial template DNA is insoluble and in a non-B-conformation. In addition to using a highly processive DNA polymerase with proofreading function, the advantage of a low temperature PCR conducted at cycling temperatures not to exceed 85˚C reduces the rate of heat-induced mutations [
The materials used for the current study were AAHS precipitates of a quadrivalent HPV vaccine in which the antigenic proteins were manufactured by a DNA recombinant technology. The viral genes encoding the L1 major capsid protein of HPV-16, HPV-18, HPV-11 and
HPV-6 were inserted into yeast cells for production of the four type-specific L1 capsid proteins used as antigens [30,31]. Based on the data presented in this report, the topological changes of the residual HPV L1 gene DNA when bound to the insoluble AAHS adjuvant seem to be genotype-related. For example, only the L1 gene DNA of the HPV-11, HPV-18 or HPV-16 has been detected, and the HPV-16 DNA needs a special method of amplification for its detection. Failure to detect HPV-6 DNA fragments may be due to a more effective removal of the HPV-6 L1 gene DNA residues from the antigen in the vaccine-manufacturing process; or the failure of detection may be caused by a special format of binding between the HPV-6 L1 gene DNA and the AAHS particles, a topological change that has turned both of its MY09 and MY11 binding sites into non-B-conformations. Since the physicochemical properties of AAHS depend on the conditions of precipitation [16-18], the dsDNA bound to the AAHS particles may vary in topology and may require different protocols for PCR amplification of different types of DNA in various vaccine batches.
Clinical trials have consistently demonstrated a higher anti-HPV-16 antibody response than an anti-HPV-18 antibody response following the use of the quadrivalent Gardasil® vaccine [32-34]. The mechanism for such a difference is poorly understood. The special conformational changes which can render the HPV-16 L1 gene DNA bound to the AAHS adjuvant less degradable in the human body than its HPV-18 or HPV-11 counterparts may provide an explanation for this difference. Codelivery of a DNA component in a protein vaccine with aluminum salts is known to stimulate a potent and multivalent immune response [35,36]. Intramuscular injection of free HPV-16 L1 plasmid DNA into BALB/C mice invariably induces a strong CD8 T cell response [
Naked HPV L1 gene DNA fragments bound to Al3+ in solid phase by ligand exchange have acquired a non-Bconformation. The resulting topological conformational changes may affect a region shared by many HPV genotypes, for example at the primer-binding site for the degenerate MY09 primer, or may affect a region of a specific genotype, for example at the degenerate MY11 primer-binding site of the HPV-16 L1 gene DNA. DNA conformational changes induced by a particulate aluminum-based adjuvant may have stabilized the residual HPV-16 L1 gene DNA fragments in an injectable vaccine and prevented their enzymatic degradation in a vaccine recipient.
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.
AAHS: Amorphous Aluminum Hydroxyphosphate Sulfate;
HPV: Human Papillomavirus;
BLAST: Basic Local Alignment Search Tool;
dNTP: Deoxynucleotide Triphosphates;
dsDNA: Double-Stranded DNA;
PCR: Polymerase Chain Reaction.