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Are HPV vaccines ‘evolution-proof’? Multilevel evolutionary ecology of human oncoviruses

Periodic Reporting for period 5 - EVOLPROOF (Are HPV vaccines ‘evolution-proof’? Multilevel evolutionary ecology of human oncoviruses)

Reporting period: 2021-09-01 to 2022-02-28

The EVOLPROOF project was conceived to develop a multi-level approach to the evolution of oncoviruses, spanning from the cellular level to the population level. It focused in particular on genital infections by human papillomaviruses (HPV), which are the most oncogenic viruses known to humans. Furthermore, safe and efficient vaccines targeting some genotypes of these DNA viruses had been implemented for approximately 10 years at the beginning of the project. This made it an opportunity to explore the selective pressure that vaccination exerts on viral populations.

This project aimed to use data and concepts from ecology and evolutionary biology to tackle issues in clinical microbiology. From a methodological point of view, it combined mathematical modelling with the setting up of a clinical study.

Overall, the project's main achievement was to set up a longitudinal cohort (PAPCLEAR) of 149 healthy young women, who were followed for a median duration of 290 days. This resulted in hundreds of gynaecological visits and thousands of biological samples. The value of the PAPCLEAR cohort resides in the density of the follow-up (clinical visits every 2 months on average and weekly self-samples) and in the quality of the data generated. Indeed, we collected detailed microbiological, immunological, and behavioural data that offer a unique opportunity to paint a dynamic picture of HPV infections, but also the vaginal microbiota. Building on evolutionary ecology theory, one goal, in particular, was to investigate the genetic diversity of the three actors (the human host, the virus, and the microbiota) to identify potential interactions.

To improve epidemiological investigations, we planned to combine this longitudinal cohort with a cross-sectional cohort of a similar size. However, due to the COVID-19 pandemic, this second part of the clinical study had to be stopped prematurely resulting in only 40 inclusions out of 150 planned. Nevertheless, the resulting overall cohort size (189 women) was sufficient to test some clinical hypotheses.

Mathematical models were developed during the project that can be used to interpret the data. All of the clinical data required for the analyses have also been collected and, in part, published. Investigations are still ongoing to characterise in detail HPV infection and vaginal microbiota dynamics.

As illustrated with the recent SARS-CoV-2 pandemic, understanding how viral infections unfold within hosts and how public health interventions, such as vaccination campaigns, impact and are impacted by virus evolution is a critical societal issue in terms of public health.
The first significant results we obtained consisted in being able to model oncovirus within-host dynamics in structured epithelial environments. This involved classical compartmental mathematical models from ecology but also experimental data from raft cultures to parameterize the model. This work led to a publication by Murall et alii in PLoS Comput Biol in 2019 and represents the foundation to analyze the virus load and immunological data collected during the PAPCLEAR study.

Another main result consisted in improving our understanding of the role of chance in the clearance of HPV infections. Using a stochastic model, we showed that the way epithelial cells infected by the virus divide can greatly affect the duration of the infection (Beneteau et alii, 2021 PLoS Comp Biol). Still, on the modelling side, we developed original approaches to analyze the diversity within infected hosts (i.e. co-infections by multiple viruses) in structured populations using network modelling (Selinger & Alizon 2021 PLoS Comput Biol). Another contribution of mathematical modelling was to investigate the multi-level evolution of oncogenic viruses (Murall & Alizon 2019 Phil Trans R Soc Lond B).

Following one of our objectives, namely to popularise an ecological approach to human infectious diseases, we wrote several review articles. Some were focused on human papillomaviruses, such as Alizon et alii (2017, Viruses), while others had a broader perspective, such as Alizon & Méthot (2018, PLoS Biology). This dissemination effort also led to the organisation of a special issue for the journal Philosophical Transactions of the Royal Society of London B entitled "Towards a multi-level and a multi-disciplinary approach to DNA oncovirus virulence".

The ongoing data analysis of the clinical study has led to preliminary results. At an epidemiological level, cross-sectional analyses of the data generated through the PAPCLEAR study have improved our understanding of the effect of vaccination on HPV diversity (Murall et alii, 2020, Vaccine). We also investigated variations in immunity profiles (Selinger et alii 2020, Immunological Research). Finally, we already reported some of the first descriptions of HPV kinetics at the 33rd International Papillomavirus Conference (IPVC).

The last category of results has to do with vaginal microbiota dynamics. The quality of the follow-ups has already led to an incidental finding regarding the use of specific types of menstrual protections (namely silicon menstrual cups) and women's health (Tessandier et alii, preprint). Additional analyses are also on the way. Finally, also regarding within-host diversity, we have been able to perform full genome virus sequencing for papillomavirus.

In terms of the dissemination of the results, the team collaborated with the gynaecology department and the STI detection clinic at the University Hospital of Montpellier since 2016. This led to the production of information leaflets on HPV infections in young adults and on vaginal microbiota and health.
The most significant progress has to do with our understanding of the dynamics of HPV genital infections in young women, and their interaction with the immune response. Hardly anything was known at the beginning of this project and we provided a first description of how HPV virus load varies over time and how it interacts with the immune response.

A second objective was to understand better the role of stochasticity (i.e. "chance") in the persistence of HPV infections. Earlier models showed that symmetric divisions of step cells matter. We further showed that the split of HPV copies between cells upon division amplifies this role.

Regarding the understanding of vaginal microbiota dynamics, we collected some of the longest longitudinal follow-up data to date, allowing us to investigate these communities' stability and diversity.

Another ambitious goal was to identify factors associated with HPV infection clearance or persistence. Investigations are on the way but some immunological markers show promising results.

Due to the premature ending of the cross-sectional cohort with the COVID-19 pandemic, our idea to investigate human genetic determinants to HPV infection clearance became impossible. However, our study can serve as the first step in setting up larger cohorts in the future.

From a more applied perspective, we were able to set up automatized pipelines to analyze detailed flow cytometry data (10 fluorescent markers) for challenging samples (cervical smears) in an unsupervised manner, which is a great asset for reproducibility and technology transfer.

Finally, our modelling work has furthered our understanding of how oncoviruses cause disease, both from a proximal standpoint (i.e. mechanistically) and from an evolutionary standpoint.
Structure of the PAPCLEAR clinical study (from Murall et al 2019)
Correlations between local cytokines concentrations (from Selinger et al 2021)
Mathematical modelling of epithelial dynamics (from Murall et al 2019)
HPV genotypes found in vaccinated and non-vaccinated women (from Murall et al 2020)