Toll-like Receptor 9 Agonists in HPV Vaccine Gardasil9

Abstract

Gardasil9 is a recombinant human papillomavirus (HPV) 9-valent vaccine, containing purified major capsid L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 re-assembled into virus-like particles (VLPs) as the active ingredients. Since the antigens are purified recombinant proteins, in theory Gardasil9 needs a potent adjuvant to generate high and sustained levels of antibodies. Historically, amorphous aluminum hydroxyphosphate sulfate (AAHS), listed as the adjuvant for Gardasil9, was known to require one or more Toll-like receptor agonists, such as the phospholipids in the recombinant hepatitis B vaccine, Recombivax HB®. However, there are no phospholipids in the purified HPV L1 proteins or in Gardasil9. But the Food and Drug Administration (FDA) reports that Gardasil4 does contain recombinant HPV L1-specific DNA fragments, and they may serve as Toll-like receptor 9 agonists in Gardasil9. The author has tested 5 samples of Gardasil9 from 4 manufacturing lots by PCR amplification with a set of degenerate primers followed by heminested PCR or by another 5 sets of non-degenerate nested PCR primers in an attempt to detect all 9 vaccine-relevant HPV type-specific L1 gene DNAs bound to AAHS in the vaccine. Sanger sequencing confirmed the presence of HPV 18, 11, 16 and 6 L1 gene DNA bound to insoluble AAHS nanoparticles, but they were unevenly distributed even within the same vaccine sample. Also, these fragments were at least partially in non-B conformations. Since no L1 gene DNA of HPV 31, 33, 45, 52, and 58 was amplified by the commonly used degenerate PCR primers, the results suggest that these may all be in non-B conformations or may have been removed as contaminants by a purification protocol. Further research is warranted to standardize the HPV DNA fragments in Gardasil which are known to be potent Toll-like receptor 9 agonists.