Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum adjuvant in the HPV vaccine Gardasil®

Detection of human papillomavirus L1 gene DNA fragments in postmortem blood and spleen after Gardasil<sup>®</sup> vaccination—A case report

“…As vaccine excipient, HPV L1 gene DNA fragments of all four genotypes, namely HPV-16, -18, -11 and -6, are expected to be present in any vaccine lot. However, previous studies have shown that only the L1 gene DNA of HPV-11 or HPV-18, or a combination of both was successfully amplified by a pair of degenerate consensus GP6/MY11 PCR primers [9], and that specially modified non-degenerate GP6/MY11 primers were needed to amplify the HPV-16 L1 gene DNA fragments bound to the insoluble fraction of the vaccine [26], indicating conformational changes taking place when naked HPV DNA is bound to the AAHS adjuvant during vaccine formulation. The topological conformational changes in the bound DNA may be genotype-related.…”

“…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][21][22][23][24][25] and for detecting residual HPV DNA fragments in the Gardasil ® vaccine [9]. Each primary PCR consisted of 1 μL of reconstituted DNA solution in molecular grade water containing about 0.5 μg human DNA, 2 μL of water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 20 μL of ready-to-use LoTemp ® PCR master mix with HiFi ® DNA polymerase (www.hifidna.com) in a total volume of 25 μL.…”

“…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 [25] and in Gardasil ® samples [9,26], generated numerous overlapping PCR product bands at agarose gel electrophoresis when the same protocol was used for testing the postmortem materials. None of such PCR products could be used for direct DNA sequencing.…”

“…HPV VLPs adsorbed to particulate amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant are exceptionally effective in eliciting production of neutralizing antibodies against HPV-16 in clinical trials [5][6][7]. The recent revelation that Gardasil ® does contain recombinant HPV L1 gene DNA fragments [8,9] which appear to be firmly bound to AAHS nanoparticles [9] may offer a plausible explanation for the high immunogenicity of Gardasil ® because co-delivery of both a DNA component and a protein component of a vaccine with aluminum phosphate salts may have the advantage of stimulating a potent and multivalent immune response [10].…”

“…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.…”