High-risk (HR) human papillomaviruses (HPVs), are the etiological agents of cervical cancer 1 . Even though infection with an HR-HPV is frequent in sexually active women, the majority of infections are resolved by the immune system in 6–12 months 2 . Only a small fraction of infections persist and can lead to cervical intraepithelial neoplasia (CIN), which may progress to cervical cancer. Factors that favour HPV persistence versus clearance, other than HPV type itself, are still poorly understood, but several studies have suggested that HPV intra-type variants could influence persistence and clinical outcome 3 4 5 6 7 8 .

A recent proposal to define major HPV variant lineages is by approximately 1.0% difference between full genomes of the same HPV type, with differences of 0.5-0.9% designated as sub-lineages 9 10 11 . Naturally occurring genetic variants of HPV 16 are common and have previously been classified into 4 major lineages; European-Asian, including the sub-lineages European (EUR), and Asian (As), African 1 (AFR1), African 2 (AFR2) and North-American/Asian-American (NA/AA). The names of the (sub)lineages derive from the geographical origin of the populations in which they were originally isolated 12 .

The most common individual polymorphism within the European lineage is T350G in the E6 oncogene, which leads to an amino acid change Leucine to Valine (L83V). Thus, the European lineage can be divided into isolates containing 350 T (EUR-350 T) or 350 G (EUR-350 G), even though this polymorphic position does not seem to define phylogenetic lineages 10 . Several studies suggest that the EUR-350 T and EUR-350 G have a different distribution among cervical cancer cases and controls and can influence persistence 3 13 14 .

The aim of this study was to assess the possible association between the genetic variants of HPV 16 and the risk of persistent HPV infection in the general population in France.

Sequencing results were obtained for a total of 142 women infected with HPV 16 with normal cytology and a valid follow-up visit. The follow-up visits ranged from minimum 4.0 months to maximum 16.0 months (median: 7.7 months, 25th percentile: 6.2 months, 75th percentile: 10.9 months). The majority of the baseline infections belonged to the European lineage (n = 131, 92.3%), with EUR-350 T and EUR-350 G accounting for 40.8% and 51.4% of the isolates, respectively. C109T (n = 3) was the only other single nucleotide polymorphism (SNP) found in more than one EUR isolate (Table 1). The number of non-EUR isolates was small (n = 11) and consisted of African-lineages and NA-lineage isolates (Table 1).

Sequence results are shown by outcome of infection: *Clearance (HPV16-negative at follow-up visit between 4–16 months) or *Persistence (HPV16-positive at follow-up visit between 4–16 months).

Abbreviations: EUR EURopean, NA North American, AFR AFRican.

0 = failed sequencing for the whole E6 gene, a shorter fragment of E6 was determined (nt. 256–400).

Of these 142 women infected with HPV 16, 86 (60.6%) showed a persistent infection at the second visit. The proportion of HPV 16 infections that persisted was slightly higher for EUR-350 T (67.2%) than EUR-350 G (56.1%) (OR = 1.6, 95% CI = 0.7-3.5), although this difference did not reach statistical significance. Adjustment for age and time to second visit did not materially alter this finding (OR = 1.6, 95% CI = 0.8-3.4) (Table 2). Among the 32 HPV 16 infections that had evidence of persistence for 12 months and more, 16 (50.0%) were EUR-350 T and 13 (40.6%) EUR-350 G (OR versus clearance =2.1, 95% CI = 0.7-5.8, data not shown).

* Clearance = HPV 16-negative at follow-up visit; Persistence = HPV 16-positive at follow-up visit.

^† aOR: Odds Ratio adjusted for age and time interval between first and follow-up visit.

Due to the rarity of non-European lineages a comparison by individual lineage could not be made. However, the proportion of all non-European lineages combined that persisted was similar to that for EUR-350 G (OR = 0.9, 95% CI = 0.2-3.4) (Table 2).

There were no statistically significant differences in the risk for persistence by HPV 16 variant lineage among women participating to the Reims HPV Primary Screening Cohort Study between 2001 and 2008. This finding, together with the distribution of the HPV 16 variants among infections at baseline, is consistent with results of a smaller and earlier study from this same cohort 14 . Nevertheless, European lineage isolates containing the polymorphism 350 T (EUR-350 T) did appear to persist more often than those containing 350 G (EUR-350 G) (OR = 1.6, 95% CI = 0.8-3.4). This is consistent with the findings of the only other population-based study of HPV 16 variant persistence in Europe: among 261 HPV 16 infections in a large cohort in Denmark, infection with EUR-350 T was associated with a significant increased risk for persistence at 2 years (OR versus EUR-350 G = 2.06, 95% CI = 1.04 - 4.25) 3 .

One weakness of the present study was the inability to make any robust reflection on viral persistence of non-EUR HPV 16 lineages, due to their rarity in the French population 4 7 15 . Furthermore, the time-interval to determine infection persistence was relatively short, driven by the use of samples from a real-life screening practice in which women with an HR-HPV infection with normal cytology were advised to return at 6–12 months.

As a consequence of viral persistence, it would be expected to find the EUR-350 T more often in cervical cancer. In accordance, Zehbe et al. showed an enrichment of EUR-350 T in invasive cervical cancer in the Czech Republic, and, to a lesser extent, in Italy. However, they also found a significant under-representation of EUR-350 T in invasive cervical cancer in Sweden 13 . This suggests that the relative oncogenic potential of HPV 16 EUR-350 T versus EUR-350 G might be population-specific 13 .

Both EUR-350 T and EUR-350 G are found commonly in precursor lesions and cervical cancers in Northern Europe, meaning that any clinical utility of variant analysis is not yet evident. Nevertheless, understanding the genetic basis of differences in persistence of HPV 16 variants may help to unravel important interactions between HPV 16 and the host immune system. This could lead to better tools to control HPV infections and its associated malignancies.

HR-HPV: High Risk Human Papillomavirus; CIN: Cervical Intra-epithelial Neoplasia; EUR: European; As: Asian; AFR: African; NA/AA: North-American / Asian-American; PCR: Polymerase Chain Reaction; SNP: Single Nucleotide Polymorphism; OR: Odds Ratio; CI: Confidence Interval.

The authors declare that they have no competing interests.

VD and CC are responsible for the Reims screening study and research databases and participated in the study design. IC performed the HPV variants testing and lead drafting of the manuscript. TG developed the HPV variant testing platform and performed variant testing. MT developed the HPV variant testing platform and participated in the data interpretation. JC participated in the data analysis and performed statistical analysis. GC participated in the study design and interpretation. SF participated in study interpretation. All authors read and approved the final manuscript.