Infection with carcinogenic or high-risk human papillomaviruses (HR-HPVs) is the main causative factor for developing cervical intraepithelial neoplasia (CIN), precursor lesions for cervical cancer (CC). CC is the first or second leading cause of cancer deaths in developing countries compared to 14th in the US [1]. Over 400,000 annual deaths occur due to CC in developing countries compared to approximately 4000/year in the US [2, 3]. In the US, 50% and 10% of CCs occur among never screened and in women who are not screened within the past 5 years, respectively, indicating a clear benefit of an organized CC screening program [4]. However, in the US, approximately 79 million women get infected with HPVs, and every year there is an estimated 14 million new cases of HPV infections [5] and approximately, 412,000 women are diagnosed with CIN annually, with an associated cost of $570 million [6]. Prevention of CIN is not only important because of the cost associated with medical care, but also because treatment of the disease has several reproductive and obstetrical related adverse outcomes [7, 8].

Quadrivalent HPV vaccine (qHPV) against HPV 16 and 18 has the ability to prevent CIN 2+ lesions or CC associated with those HPV genotypes [9, 10]. However, the fact that a substantial proportion of CIN 2+ lesions are caused by HR-HPV types other than HPV 16 or 18 has begun to be appreciated [11]. The inclusion of additional HR-HPV genotypes (31, 33, 45, 52, and 58) in the 9-valent HPV vaccine (9VHPV) is expected to have better CIN preventive effects, but even this vaccine does not include six HR-HPV genotypes (35, 39, 51, 56, 59, 68). Previous studies have shown that these non-vaccine HPV genotypes are associated with CC risk [12]. Our studies have shown that non-vaccine HPV genotype associated CIN 2+ lesions may have similar malignant potential compared to such lesions that are associated with HPV genotypes that are included in vaccines [13]. No previous studies, however, have reported the determinants of CIN 2+ lesions that are unlikely to be preventable by HPV vaccines. We previously reported that among women who tested positive for any one of the 13 HR-HPVs, those with supraphysiologic concentrations of plasma folate (> 19.8 ng/ml) and sufficient plasma vitamin B₁₂ (> 200.6 pg/ml) had 70% reduced risk of developing CIN 2+ due to their known cancer protective functions [14]. Carotenes are thought to play a role in reducing the risk of cancer in the head and neck, another HR-HPV associated cancer [15].

Based on this background, the purpose of this study was to: 1) identify the most prevalent non-vaccine HPV genotypes detected in CIN 2+ lesions (cases) compared to ≤ CIN 1 (non-cases); and 2) determine whether plasma concentrations of folate, vitamin B₁₂ and carotene and socio-demographic, lifestyle related CIN risk factors alter the risk of being diagnosed with nonvaccine HPV related CIN 2+.

Prophylactic HPV vaccines hold promise to prevent the development of CIN 2+ due to HPV genotypes that are included in vaccines, but a demonstrable reduction of the burden of CC, the main goal of prophylactic HPV vaccines, is expected to take several decades. In addition, women will continue to develop CIN 2+ lesions due to HR-HPV genotypes that are not included in such vaccines and those lesions will continue to progress to CC. Therefore, vaccine approach is likely to decrease but not entirely prevent the development of HPV-related CCs for the foreseeable future. Therefore, we envision that a continuation of tailored screening programs combined with lifestyle changes and other non-vaccine-based approaches will be necessary to manage the risk of developing HPV-related cancers in the post-vaccine era. Thirteen HPV genotypes are considered to be high risk, namely, HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. It is generally accepted that HPV 16 and 18 are the most carcinogenic, but studies report that HPV genotypes such as HPV 33, 35 and 45 have similar carcinogenic potential to that of HPV 16 and 18 via producing similar concentrations of mRNA of E6/E7 oncoproteins [17, 18, 19] and those interfere with the normal growth regulatory functions of the host cellular proteins P53 and RB that favor malignant transformation [20, 21, 22]. Even though these observations indicate that women infected with other HR-HPV genotypes, even in the absence of HPV genotypes included in HPV vaccines may develop lesions with similar malignant potential, no studies have identified factors that alter the risk of being diagnosed with non-vaccine HPV related CIN 2+ or the most prevalent non-vaccine HPV genotypes detected in such lesions. Studies in the US and in other countries have demonstrated reductions in CIN 2+ lesions attributable to HR-HPV genotypes targeted by qHPV vaccine [23, 24, 25]. Studies have also estimated that if 9VHPV vaccination programs are effectively implemented, the majority of CIN 2+ due to those HPV genotypes could be prevented [26, 27]. However, the 9VHPV vaccine failed to prevent infection or the development of precancerous lesions in the cervix, vulvar or vagina related to HPV types beyond the nine types covered by this vaccine [31]. Since the magnitude of CIN burden related to non-vaccine HPV genotypes or their determinants is largely unknown, in this study, we addressed these concerns by utilizing a population of women who were are characterized for demographic, lifestyle related CIN risk factors, plasma concentrations of folate, vitamin B₁₂ and carotene, HPV status and histologically confirmed CIN diagnoses. We observed that > 50% of our study population tested positive for HPV genotypes that are not included in qHPV and 15% and 7% of them had CIN 2 or CIN 3, respectively. The percentage of women who tested positive for HR-HPV genotypes that are not included in the 9VHPV vaccine was lower (22%), but 13% and 5% of them had CIN 2 and CIN 3, respectively. These observations indicate that CC risk in women vaccinated with current vaccines cannot be ignored. Further, the cumulative incidence of such lesions could be higher in this population over time since 78% and 82% of women diagnosed with ≤ CIN 1 who are at risk of progressing to CIN 2+ are also positive for HR-HPV genotypes that are not included in qHPV and 9VHPV vaccines, respectively. Further, the prevalence of some of these HPV genotypes such as HPV 35 was higher than 60% and the prevalence was significantly higher in CIN 2 or 3 compared to CIN 1 lesions, especially in AA women. Since AAs and CAs are the most prevalent racial groups in the USA and there are cultural differences with regard to dietary factors between these racial groups, it is important to document racial differences in CIN 2+ risk related to specific HPV genotypes. We observed that women who receive qHPV or 9VHPV vaccines are more likely to develop CIN 2 rather than CIN 3. Even though the severity is lower, CIN 2 lesions left under observation without treatment according to the current American College of Obstetricians and Gynecologists (ACOG) guidelines in women < 25 years of age may transform to CIN 3 by the time they reached screening age. Treatment of CIN 2 in older women results in adverse reproductive and gynecological outcomes indicating the importance of their prevention in vaccinated women, irrespective of their age. We observed that improving vitamin B₁₂ status or its supplementation could serve as a non-vaccine-based approach to reduce the risk of developing such lesions. In contrast to our previous findings reported with women enrolled during at the beginning of the US folic acid fortification period (1996-1998), we did not observe a significant association between lower folate status and higher risk of CIN 2+ [28]. This could be due to the fact that plasma folate concentrations had continued to increase in US over time due to the folate fortification program. We previously reported that in a cohort of women enrolled after 2004, a few years of exposure to folate fortified food, having sufficient vitamin B₁₂ plays a significant role in altering the risk of developing CIN 2+ [14]. Since there was no vitamin B₁₂ fortification program in the US, approximately, one third of women who are at risk for CIN 2+ have inadequate concentrations of vitamin B₁₂ (< 350 pg/mL). In developing countries such as India, however, both folate and vitamin B₁₂ could play a role in reducing CIN 2+ risk as only 2% achieve supra-physiologic concentrations of folate and 66% of women have inadequate vitamin B₁₂ concentrations [29]. These observations suggest that improving the status of both folate and vitamin B₁₂ by dietary means or their supplementation could serve as an effective non-vaccine-based approach to reduce the risk of developing such lesions. Mechanistically, folate is likely to be associated with a lower risk of CIN 2+ in several ways. Folate deficiency leads to DNA instability (DNA and chromosomal damage), risk factors for cancer, by several mechanisms including uracil misincorporation, impaired DNA excision repair, and suboptimal cellular DNA repair capacity [30. 31]. Higher folate minimizes these DNA and chromosomal changes [32]. Intervention studies in humans taking folate supplements have shown that DNA damage in peripheral blood cells is minimized when plasma concentrations of folate is high [30]. Further, DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro [29]. Folate deficiency also modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes [33]. Folate’s influence on HPV integration is also a possible explanation since common chromosomal fragile sites that are sensitive to folate deficiency have been shown to coincide with sites of HPV integration, a key event in HPV carcinogenesis [34]. Further, our previous studies have showed that a lower degree of HPV methylation in the presence of lower plasma concentrations of folate and vitamin B₁₂ influence the biology of specific HPV genotypes and the likelihood of being diagnosed with CIN 2+ [35], indicating the importance of both folate and vitamin B₁₂ to prevent the development of CIN 2+. We also observed that AA women and smokers who will be given qHPV vaccine may need regular follow up and advice on smoking cessation suggesting that tailored preventive measures based on ethnicity and adverse health habits are important. In this study, we noted that none of the known risk factors evaluated was significantly associated with the risk of developing CIN 3. However, since the magnitude of the association was similar for some risk factors such as smoking, the smaller sample size may explain these non-significant results. It is also possible that other factors that are not examined such as methylation of HPV or their integration status could be contributing to the development of such CIN lesions. Collectively, our results demonstrated the importance of continuation of tailored screening programs combined with lifestyle changes/other non-vaccines-based approaches to manage the residual risk of developing HPV related cancers in the post-HPV vaccination era as the prevalence of some HPV genotypes that are not included are frequently found in this population. Further, since the incidence of HPV-associated pre-cancers and cancers also continues to increase at several anatomical sites other than the cervix (oropharynx, anus, vagina, vulva) [36] in the post-HPV vaccination era, it is important to study and extend similar preventive approaches to control the risk of developing cancers in those organs. Our study approach will form the foundation for such studies. In this study, we assumed that women who tested negative for qHPV or 9VHPV genotypes could have received those vaccines and had 100% efficacy in preventing infections with those vaccine HPV genotypes. Even though this assumption can be viewed as a limitation, it is important to acknowledge that HPV vaccine studies are in general are conducted in younger females who are naive to HPV infections and therefore cervical cells to test for HPV infections or biopsies to evaluate CIN diagnoses are rarely collected in these studies and therefore it is not possible to test the aims of our study in those populations. Our approach will form the foundation to design post vaccination era studies in the future to test the significance of tailored screening programs combined with lifestyle changes/other non-vaccines-based approaches to manage the residual risk of developing HPV related cancers in vaccinated women. Since 2016, only 9VHPV vaccine is used in the US. However, there is no Advisory Committee on Immunization Practices (ACIP) recommendation regarding additional 9VHPV vaccine doses for people who have completed the vaccine series with the qHPV vaccine other than 9VHPV vaccine may be used to continue or complete a vaccination series started with qHPV vaccine [37, 38, 39, 40]. Therefore, both groups of women, negative for qHPV and 9V HPV genotypes will exist in the US post HPV vaccination era. Since in developing countries only the qHPV vaccine is used currently, vaccinated women will only be negative for qHPV genotypes.

The current HPV vaccine approach is likely to decrease but not prevent the development of cervical precancerous lesions entirely in a significant proportion of women. Continuation of screening programs combined with lifestyle changes and other non-vaccine-based approaches are needed to manage the risk of developing HPV-related cancers in vaccinated women. We identify vitamin B₁₂ status and smoking as independent modifiable factors and ethnicity as a factor that needs attention to reduce the risk of developing CIN 2+ in the post vaccination era. Larger studies are needed to confirm the significance of other factors such as educational status, parity and the number of lifetime partners that reached borderline significance in relation to CIN 2 or 3 in our study. Since the current study is a cross-sectional study where risk factors and plasma concentrations micronutrients were assessed at a single time point, the temporality of the associations or causal relations cannot be assessed in our study. Verification of our results in longitudinal studies and their reproducibility in other populations exposed to HPV infections and/ vaccines will increase the scientific validity of our findings.