Quote this article as:

L Teblick, S Van Keer, A Vorsters (March 2022). First-void urine as liquid biopsy for HPV vaccine-induced antibody detection. www.HPVWorld.com, 191

Appropriate and reliable biological samples are critical for advancing public and personal health. Having a (liquid) sample that is non-invasive and can be self-collected has significant advantages within the field of immunology and carcinology¹. First-void urine is an excellent example of a liquid biopsy that is highly acceptable, non-invasive and can be self-collected at home^2-4. As genital tract secretions are washed away at the beginning of urination, first-void urine sampling has considerable potential for human papillomavirus (HPV) research^5-7. Several studies have demonstrated that first-void urine is a suitable sample to detect HPV DNA and that the DNA remains stable by using a preservative, allowing future use for cervical screening that now is primarily carried out using a clinician-collected cervical smear³. Monitoring immune response in clinical trials and vaccination programs is based on serum samples to assess anti-HPV vaccine-induced antibodies. Since immunoglobulin G (IgG) antibodies are widely present in genital tract secretions, we hypothesised that IgG antibodies could be detectable using first-void urine sampling⁸. Indeed, our latest research confirmed that first-void urine contains sufficient genital tract secretions to detect vaccine-induced anti-HPV antibodies^9,10.

First-void urine sampling can be used to monitor both disease and immune response, making it an ideal sample for surveillance and follow-up of vaccine trials, combining the capacity of a clinician-taken cervical smear and a blood sample (Figure 1). In addition, the suitability of first-void urine for at-home sampling and its ethical acceptability for girls too young for gynaecological examination are major advantages for long-term monitoring of vaccine impact¹¹.




In 2018, a proof-of-concept trial for the detection of anti-HPV antibodies in first-void urine was set up where we investigated the presence of anti-HPV antibodies in first-void urine⁹. Within this study, 57 women (aged 19-26 years) provided paired first-void urine and blood samples. These women were divided in two cohorts; one cohort vaccinated during adolescence (n=38) and one cohort non-vaccinated (n=19). Samples were tested using two HPV immunoassays: glutathione S‐transferase (GST)‐L1‐based immunoassay (GST‐L1‐MIA, DKFZ, Germany) and multiplex L1/L2 virus‐like particles (VLP)‐based ELISA (M4ELISA, CDC, Atlanta)^9,10. The results showed statistically significant correlations between anti-HPV antibody concentrations in first-void urine and serum samples (Figure 2). In addition, we detected significantly higher anti-HPV antibody concentrations in vaccinated compared to unvaccinated women for both sample types (Figure 3). This study proved the feasibility of detecting anti-HPV antibodies after bivalent and quadrivalent HPV vaccination. Currently, we are conducting a second clinical trial where we collect first-void urine samples before nonavalent vaccination, seven months after the first dose and four years after the first dose. Here, we aim to provide evidence that first-void urine can be used for long-term follow-up trials (preliminary results presented at Eurogin 2020 and IPVC 2021). Additional ongoing research includes optimising the developed techniques and normalisation and validation using larger cohorts.









In addition, using a first-void urine sample offers more opportunities within HPV-related research. Although we expect no therapeutic effects of the currently available prophylactic HPV vaccines, the neutralising antibodies induced after vaccination may interact with newly produced virions. This interaction may affect existing HPV infections by preventing virions from spreading to other sites in the genital tract (autoinoculation) and preventing transmission to the sexual partner¹². This biologically plausible hypothesis still needs further molecular proof and is currently being investigated at our research centre. Since first-void urine sampling easily captures genital secretions containing intact functional antibodies, we also expect it to contain intact HPV virions (Figure 1)¹³. Investigating if the vaccine-induced anti-HPV antibodies neutralise intact HPV virions and therefore could avoid autoinoculation and transmission to another person remains challenging. However, if confirmed, this will substantially impact how we can optimally model, assess and implement HPV vaccination programs since there is still reluctance to vaccinate (young) women post-sexual debut or women diagnosed with an HPV infection.

In summary, first-void urine has the potential to become the ideal liquid genital tract biopsy for HPV-related research. Using only one non-invasive sample collected at home for both follow-up of infection and vaccination creates novel and promising opportunities for disease and vaccine monitoring in large epidemiological studies. In addition, using a first-void urine sample will aid in our search to define a correlate of protection.


ACKNOWLEDGEMENTS
Part of this work performed by LT, SVK and AV was supported by the Industrial Research Fund of the University of Antwerp, Belgium (Grant No. PS ID 32387) and a Merck Investigator Study Program (Grant No. IIS #59401). SVK is supported by a junior postdoctoral fellowship of the Research Foundation – Flanders (FWO), Belgium (Grant No. 1240220N). AV participated in advisory board meetings of Merck. Participation fees are paid directly to an educational fund held by the University of Antwerp. AV works partly for the executive secretariat of the HPV prevention and control board. The secretariat is seated at the Centre for the Evaluation of Vaccination (CEV) of the University of Antwerp, where it has the infrastructure and administrative services of the University at its disposal. The HPV prevention and control board is supported by unrestricted grants from the industry (including GlaxoSmithKline Biologicals, Sanofi Pasteur MSD, Merck, Abbott and Hologic), several universities in Europe and other institutions. AV is co-founder of Novosanis (Belgium), a spin-off company of the University of Antwerp, and was minority shareholder until January 2019.


References

1. Siravegna G et al. How liquid biopsies can change clinical practice in oncology. Annals of Oncology vol. 30 1580–1590 (2019). Available from: https://doi.org/10.1093/annonc/mdz227

2. van Keer S et al. First-void urine: A potential biomarker source for triage of high-risk human papillomavirus infected women. European Journal of Obstetrics & Gynecology and Reproductive Biology 216, 1–11 (2017). Available from: https://doi.org/10.1016/j.ejogrb.2017.06.036

3. Vorsters A et al. Optimization of HPV DNA detection in urine by improving collection, storage, and extraction. European Journal of Clinical Microbiology & Infectious Diseases 33, 2005–2014 (2014). Available from: https://doi.org/10.1007/s10096-014-2147-2

4. Pattyn J et al. HPV DNA detection in urine samples of women: ‘an efficacious and accurate alternative to cervical samples?’ Expert Review of Anti-Infective Therapy vol. 17 755–757 (2019). Available from: https://doi.org/10.1080/14787210.2019.1668776

5. Augustus E et al. The art of obtaining a high yield of cell-free DNA from urine. PLoS ONE 15, (2020). Available from: https://doi.org/10.1371/journal.pone.0231058

6. Vorsters A et al. Urine testing for HPV: Rationale for using first void. BMJ (Online) 349, 6252 (2014). Available from: https://doi.org/10.1136/bmj.g6252

7. Pathak N et al. Accuracy of urinary human papillomavirus testing for presence of cervical HPV: Systematic review and meta-analysis. BMJ (Online) 349, (2014). Available from: https://doi.org/10.1136/bmj.g5264

8. Pattyn J et al. A. Non-invasive Assessment of Vaccine-Induced HPV Antibodies via First-Void Urine. Frontiers in Immunology 11, (2020). Available from: https://doi.org/10.3389/fimmu.2020.01657

9. van Keer S et al. First-void urine as a non-invasive liquid biopsy source to detect vaccine-induced human papillomavirus antibodies originating from cervicovaginal secretions. Journal of Clinical Virology 117, 11–18 (2019). Available from: https://doi.org/10.1016/j.jcv.2019.05.004

10. Pattyn J et al. Comparison of a VLP-based and GST-L1-based multiplex immunoassay to detect vaccine-induced HPV-specific antibodies in first-void urine. Journal of Medical Virology 92, 3774–3783 (2020). Available from: https://doi.org/10.1002/jmv.25841

11. Franceschi S et al. Options for design of real-world impact studies of single-dose vaccine schedules. Vaccine 36, 4816–4822 (2018). Available from: https://doi.org/10.1016/j.vaccine.2018.02.002

12. Schiller JT, Davies P. Delivering on the promise: HPV vaccines and cervical cancer. Nature Reviews Microbiology 2, 343–347 (2004). Available from: https://doi.org/10.1038/nrmicro867

13. Vorsters A et al. HPV vaccination: Are we overlooking additional opportunities to control HPV infection and transmission? International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 88, 110–112 (2019). Available from: https://doi.org/10.1016/j.ijid.2019.09.006