# The magnitude and durability of neutralizing antibody responses to human papillomavirus vaccine do not depend on DNA sensing pathways

## TL;DR

This study finds that DNA sensing pathways are not needed for strong and lasting antibody responses to the 9-valent HPV vaccine.

## Contribution

The study shows that DNA sensing pathways do not influence the magnitude or durability of HPV vaccine-induced neutralizing antibodies.

## Key findings

- 9vHPV vaccine contains low copy numbers of L1 DNA but not other HPV types.
- Neutralizing antibody responses to 9vHPV are not dependent on DNA sensing pathways like TLR9, cGAS, or STING.
- Plasma cell responses to HPV16 and HPV18 are consistent across different mouse strains.

## Abstract

How human papillomavirus (HPV) vaccines elicit robust and enduring neutralizing antibody (nAb) responses is unknown, yet such information is valuable to vaccinology. In addition to the major capsid protein, L1, the 4-valent HPV vaccine reportedly also contains recombinant L1 DNA. Nucleic acids in vaccines enhance antibody responses and are employed as adjuvants, but whether L1 DNA enhances HPV vaccine antibody responses is understudied. We tested whether 9-valent HPV (9vHPV) vaccine lots contained L1 DNA and if peak or long-term 9vHPV vaccine-elicited nAb responses were dependent on DNA sensors Toll-like receptor 9 (TLR9), AIM2, or cGAS or on STING or MyD88, the adaptors for cGAS and TLR9. To quantify L1 DNA, we extracted total nucleic acid per 9vHPV dose and applied quantitative PCR to amplify HPV6/11/16/18/31/33/45/52/58 L1 sequences. Wild-type mice (C57BL/6J) or mice deficient in DNA sensing pathways (TLR9-/-, AIM2-/-, cGAS-/-, STINGgt/gt, MyD88-/-) were administered 9vHPV vaccine or equivalent adjuvant only at 0, 4, and 12 weeks. We measured serum HPV16 and HPV18 nAb titers at 16, 26, 40, and 53–56 weeks and HPV16- and HPV18-specific bone marrow plasma cell responses at 53–56 weeks. We detected 5–44 and 104–351 copies of HPV6 and HPV18 L1 DNA per 9vHPV dose, but no other types (n = 3 lots). We found no consistent difference in geometric mean nAb titers between mice strains that received 9vHPV at any time point tested or in median HPV-specific plasma cell frequencies. Thus, we conclude the magnitude and durability of nAb responses to 9vHPV vaccination do not depend on DNA sensing pathways.

How the highly effective human papillomavirus (HPV) vaccines elicit strong and long-lasting antibody responses is unknown, yet such knowledge is valuable to vaccine development. The HPV vaccines are comprised of virus-like particles assembled from the HPV major capsid protein, L1; an adjuvant; and reportedly also residual recombinant L1 DNA as a result of the manufacturing process. We tested whether the currently available 9-valent HPV (9vHPV) vaccine contained L1 DNA and if 9vHPV vaccine-elicited antibody responses were dependent on DNA sensing pathways of the innate immune system in vivo. We confirmed separate lots of the 9vHPV vaccine contained low copy numbers of L1 DNA, but found 9vHPV vaccine-generated antibody responses were not dependent on DNA sensing. This study has thus refined our understanding of potential mechanisms underlying the strong and long-lasting antibody responses to HPV vaccination.