Distribution of human papillomavirus type 16 variants in human immunodeficiency virus type 1-positive and -negative women

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1 Journal of General Virology (2004), 85, DOI /vir Short Communication Distribution of human papillomavirus type 16 variants in human immunodeficiency virus type 1-positive and -negative women Anil K. Chaturvedi, 1 Joeli A. Brinkman, 2 Ann M. Gaffga, 2 Jeanne Dumestre, 2 Rebecca A. Clark, 4 Patricia S. Braly, 4 Kathleen Dunlap, 4 Patricia J. Kissinger 1 and Michael E. Hagensee 2,3 Correspondence Michael E. Hagensee mhagen@lsuhsc.edu 1 Department of Epidemiology, Tulane University School of Public Health and Tropical Medicine, 1440 Canal Street, New Orleans, LA 70112, USA 2,3,4 Departments of Microbiology 2, Medicine 3 and Obstetrics and Gynecology 4, Louisiana State University Health Sciences Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA Received 2 October 2003 Accepted 17 January 2004 The prevalence of human papillomavirus type 16 E6 variant lineages was characterized in a cross-sectional study of 24 human immunodeficiency virus type 1 (HIV)-positive and 33 HIV-negative women in New Orleans. The European prototype was the predominant variant in the HIV-negative women (39?4 %), while in the HIV-positive women the European 350G variant was predominant (29?1 %). In exact logistic regression models, HIV-positive women were significantly more likely to harbour any variant with a nucleotide G-350 mutation compared with HIV-negative women [58?3 % vs 21?1 %; adjusted odds ratio (AOR)=6?28, 95 % confidence interval (CI)=1?19 46?54]. Models also revealed a trend towards increased prevalence of Asian American lineage in HIV-positive women compared with HIV-negative women (25?0% vs 6?0 %; AOR=6?35, 95 % CI=0?77 84?97). No association was observed between any variant and cytology or CD4 cell counts or HIV-1 viral loads. These observations reflect a difference in the distribution of HPV-16 variants among HIV-positive and -negative women, indicating that HIV-positive status may lead to increased prevalence of a subset of variants. Human papillomavirus (HPV) type 16 is the most prevalent genital HPV type and accounts for ~50 % of cervical cancer cases worldwide (Bosch et al., 1995; Yamada et al., 1997; Munoz et al., 2003). In spite of high prevalence and robust associations with cervical cancer, the majority of HPV-16 infections are transient and only a fraction progress to develop pre-invasive and invasive lesions (Walboomers et al., 1999), underscoring the interplay of additional factors in development of cervical cancer (Munoz et al., 1992; Hildesheim et al., 2001a; Moreno et al., 1995) such as behavioural (smoking, parity, oral contraceptive use) (Castellsague et al., 2002), host genetic (HLA phenotypes and p53 polymorphisms) (Giannoudis & Herrington, 2001; Hildesheim & Wang, 2002; Maciag & Villa, 1999) and viral factors (HPV genotypes, intratype variants and viral load) (Hildesheim & Wang, 2002; Londesborough et al., 1996; Lorincz et al., 1992, 2002). HPV variants are defined by 0 2 % sequence heterogeneity in the L1, L2 and E6 genes (Yamada et al., 1995). HPV-16 intratype variants have been shown in molecular studies to have altered oncogenic potential (Stoppler et al., 1996). Several epidemiological studies have corroborated this difference in oncogenic potential, specifically for the non-european variants (Hildesheim et al., 2001b; Xi et al., 1997, 1998, 2002), European 350G variants (Andersson et al., 2000; Zehbe et al., 1998, 2001; Kammer et al., 2002) and the Asian American lineages (Berumen et al., 2001). This difference in oncogenic potential could be attributed to one or a combination of the following: (i) increased infectivity and/or increased virus persistence; (ii) increased ability to cause immortalization of cervical epithelial cells; or (iii) increased ability to escape immune surveillance. The cell-mediated immune system is believed to be central to the control of HPV infection, as evidenced by an increase in the incidence, prevalence and persistence of HPV infections in immunocompromised individuals, particularly human immunodeficiency virus type 1 (HIV)-positive women (Minkoff et al., 1998; Ahdieh et al., 2001; Sun et al., 1997; Palefsky et al., 1999; Vernon et al., 1994). Little is known regarding the immune control of HPV variants; in addition, few studies have compared the prevalence of HPV variants in HIV-positive and -negative subjects (Perez-Gallego et al., 2001; Icenogle et al., 1992). Comparison of variant prevalence among HIV-positive and -negative G 2004 SGM Printed in Great Britain 1237

2 A. K. Chaturvedi and others women and across CD4 T cell strata may provide further insight into the possible role of cellular immunity on the prevalence of variants. Two-hundred and thirty-seven HIV-positive women attending the HIV outpatient clinic and 622 HIV-negative women attending the Colposcopy clinic in the Medical Center of New Orleans (MCLNO) associated with the Louisiana State University Health Sciences Center (LSUHSC) participated in this cross-sectional study. DNA from cervical/ vaginal swabs was extracted (Ting & Manos, 1990) and HPV DNA was detected using the PGMY09/11 biotinylated primer system (Gravitt et al., 1998). Genotyping was performed by reverse line blot hybridization (Roche Molecular Systems) (Gravitt et al., 1998). HPV-16-positive specimens, 24 from HIV-positive and 33 from HIV-negative subjects, were subjected to PCR using HPV-16 E6 type-specific primers (Zehbe et al., 1998) and extension products were subjected to cycle sequencing using an ABI Prism 377 automated DNA sequencer (Applied Biosystems). The HPV-16 E6-specific PCR primers were used as sequencing primers. For n=29 samples, PCR and sequencing of the opposite strand could not be repeated owing to specimen inadequacy. All sequences with 100 % similarity to the reference sequence and sequences showing 1 2 nt changes that did not fall into signature patterns of any variant lineages were assigned as prototype European 350T sequences. CD4 T cell counts and HIV-1 RNA viral load information were collected from the Adult Spectrum of Diseases (ASD) database. When CD4 counts or HIV-1 RNA viral loads were not available for the exact date of study participation, results were collected from the nearest 3 month window before or after the date of study participation. Exact logistic regression (Mehta & Patel, 1995) was used to derive unadjusted and age-adjusted (age categorized as a binary variable, 25 and >25) odds ratios (OR) and 95 % confidence intervals (CI) for prevalence of each variant lineage compared with European 350T prototype between HIV-positive and -negative women. All analyses were two-sided using an alpha of 0?05; analyses were completed in SAS version 8.2 for Windows and adjustments for multiple comparisons were not performed. The results in this paper were presented in part at the 20th International Papillomavirus Conference, 4 9 October, 2002, Paris, France. Informed consent was obtained from all participants/patients and all procedures followed in conducting the clinical research were in accordance with the Institutional Review Board at the Louisiana State University Health Sciences Center, New Orleans. Twenty-four (10?1 %) subjects in the HIV-positive cohort and 33 (5?3 %) subjects in the HIV-negative cohort were positive for HPV-16. The demographic characteristics of the HPV-16-positive HIV-positive and -negative cohorts are presented in Table 1. HIV-positive women were significantly older than HIV-negative women (x 2 P=0?014) and both the HIV-positive and -negative cohorts were predominantly African American. No significant differences Table 1. Demographic characteristics of the HPV-16- positive cohort (n=57) Percentages may not add up to 100 % owing to missing values. Characteristic HIV-positive (n=24) (%) HIV-negative (n=33) (%) x 2 P value Age 0?014 Mean±SD 32?83±8?23 25?75±6? (25?0) 19 (57?6) >25 18 (75?0) 14 (42?4) Race* 0?708 African American 18 (75?0) 26 (78?8) Caucasian 4 (16?6) 4 (12?1) Other 1 (4?2) 3 (9?1) Pap smear statusd 0?249 Normal 4 (16?7) 15 (45?5) ASCUS 6 (25?0) 7 (21?2) LGSIL 7 (29?2) 6 (18?2) HGSIL 3 (12?5) 4 (12?1) CD4 cell counts mm 3 d <200 9 (37?5) NA > (54?2) HIV-1 RNA viral load ml 1 d < (58?3) NA > (29?2) *Ethnicity was missing for one subject in the HIV-positive cohort. DPap smear data were missing for four HIV-positive and one HIVnegative subjects. dcd4 counts were missing for two subjects and HIV-1 RNA viral loads were missing for three subjects. NA, Not applicable. were observed in the Pap smear status between the HIVpositive and -negative women (P=0?249). Prevalence of variant lineages in the HIV-positive and -negative cohorts and unadjusted and age-adjusted exact OR and 95 % CI comparing prevalence of each variant lineage to the European 350T prototype between HIV-positive and -negative women are shown in Table 2. The most prevalent variant in the HIV-positive cohort was the European 350G lineage (29?1 %), while the European 350T prototype was the most prevalent lineage in the HIV-negative cohort (39?4 %). In unadjusted logistic regression analyses with exact inference, the prevalence of Asian American variants was higher, though not statistically significant, in the HIV-positive women (25?0 % vs 6?0 %, exact P value=0?068). When comparisons were performed combining all variant lineages with a TRG mutation at nt 350, HIV-positive women were significantly more likely to harbour an nt 350 G mutation as compared with HIV-negative women (58?3% vs 21?2 %, exact P value=0?034). Exact logistic regression analyses adjusting for age revealed a trend towards increased prevalence of Asian American variants (see Table 2) in HIV-positive women compared 1238 Journal of General Virology 85

3 HPV-16 E6 variants in HIV-positive/negative women Table 2. Prevalence of HPV-16 E6 variant lineages in HIV-positive and -negative women (n=57) Values in bold are statistically significant at P<0?05. Variant HIV-positive (n=24) (%) HIV-negative (n=33) (%) Unadjusted exact OR (95 % CI) Age-adjusted exact OR (95 % CI) European 350T prototype* 5 (20?8) 13 (39?4) 1?00 1?00 European 350G 7 (29?1) 5 (15?2) 3?47 (0?61 22?37) 4?80 (0?67 58?99) European 131G 1 (4?2) 1 (3?0) 1?28 (0?01 30?28) 2?21 (0?02 192?09) African-1 3 (12?5) 5 (15?2) 1?53 (0?17 12?15) 0?99 (0?07 8?72) African-2 1 (4?2) 5 (15?2) 0?53 (0?01 6?90) 1?40 (0?01 109?89) Asian American 6 (25?0) 2 (6?0) 7?11 (0?89 95?55) 6?35 (0?77 84?97) UncharacterizedD 1(4?2) 2 (6?0) 2?46 (0?02 219?47) 2?44 (0?03 196?19) Total 24 (100?0) 33 (100?0) Any variantd 19 (79?2) 20 (60?6) 2?55 (0?67 11?15) 2?80 (0?67 13?58) Any 350G variant 14 (58?3) 7 (21?2) 4?96 (1?10 26?00) 6?28 (1?19 46?54) *Includes one specimen with an nt 90 ARC mutation and one specimen with an nt 528 CRT in the HIVpositive women, and two specimens with an nt 83 ART, two specimens with an nt 90 ARC and one specimen with an nt 187 ARG mutation in the HIV-negative women. DIncludes specimens with indeterminate variant lineages. dincludes all variant lineages. Includes European 350G, Asian American variant lineages and one G-131 variant in the HIV-positive women. with HIV -negative women (exact P value=0?097) and significantly higher prevalence of variants with a mutation at nt 350 in HIV-positive women (exact P value=0?026). In addition, comparisons were performed categorizing the variant lineages as (i) prototype-like (including European 350T) and any variant (including European 350G, European G131T/G, African-1, African-2 and Asian American variants); and (ii) European variants (including European 350T, European 350G, European G131T/G variants) and non-european variants (African-1, African-2 and Asian American variants). No significant differences were observed between the HIV-positive and -negative women using either of these classifications. Similar comparisons performed in the HIV-positive women stratifying by CD4 cell counts and HIV-1 RNA viral loads revealed no significant differences between either CD4 counts or viral loads and prevalence of any variant lineage. The most prevalent lineage in women with squamous intraepithelial lesions was the European 350G (40?0 %) in the HIV-positive women and the European 350T prototype in the HIVnegative women (50?0 %). No significant differences were observed in the prevalence of any variant lineage compared with the prevalence of prototype in women with cytological abnormalities in either cohort. The prevalence of non-european variant lineages in the cohort (38?5 %) was relatively high compared with other studies conducted in the USA (Yamada et al., 1997; Xi et al., 1997, 1998, 2002; Da Costa et al., 2002). Yamada et al. (1997) reported an 8 % prevalence of non-european lineages in 30 cervical cancer cases from the USA. Similarly, Xi et al. (1997) reported non-european lineage rates of 21 % and 14 % in university students and women from sexually transmitted disease clinics, respectively. More recently, Da Costa et al. (2002) in a study of anal neoplasia reported a 33 % prevalence of European variants in a cohort of 39 HIVpositive women. Considering the predominant African American nature of the study population, this relatively high prevalence of non-european variants is consistent with studies showing increased non-european lineages in non-white populations (Xi et al., 2002). Few studies have assessed the prevalence of HPV-16 variants in HIV-positive subjects. Icenogle et al. (1992) sequenced the L1 gene of three HPV-16 isolates from Kinshasa, Zaire, and reported prevalence of similar variants in HIV-positive and -negative specimens. A recent study from Spain comparing 48 HIV-negative and 13 HIV-positive subjects reported no difference in the prevalence of nt 350 G variants by HIV status (Perez-Gallego et al., 2001). In contrast, in this paper we have reported an increased prevalence of variant lineages with an nt 350 G mutation in HIV-positive women and a trend towards increased prevalence of Asian American variants in HIV-positive women. The contrasting results probably arise from the differences in geographic location and sample sizes. Moreover, both previous studies recruited only women with cervical intraepithelial neoplasia. One explanation for the observed differences between HIV-positive and -negative women could be a clustering of certain variants in the HIV-positive cohort owing to sexual networking. An increase in the number of sequence variants has been reported in women with multiple sex partners who are at a high risk of acquiring sexually transmitted diseases as compared with monogamous women (van Belkum et al., 1995). We could not adjust

4 A. K. Chaturvedi and others the associations for either number of recent sex partners or number of lifetime sex partners in this study since this information was available only for the HIV-positive women. Increased persistence of HPV infections in HIV-positive women has been well documented (Sun et al., 1997) and HPV-16 variants with a mutation at nt 350 have also been associated with increased persistence of infection (Londesborough et al., 1996). If, indeed, variants with a mutation at nt 350 increase the persistence of infections, this may be further enhanced owing to HIV-induced immune loss. Thus, it would be predicted that the nt 350 variants in the HIV-positive women represent persistent infections. As a result of the use of prevalent infections and not incident infections in this study, the duration of infection could not be assessed. The observation of increased prevalence of variants with an nt 350 mutation in the current study could be explained by the increase in cervical dysplasia previously reported in HIV-positive women. However, in the current study, the prevalence of cervical dysplasia was not significantly different between the HIV-positive and -negative women. Ellis et al. (1995) reported that a mutation at nt 131 of HPV-16 E6 resulted in a change in T cell responses. Similarly, a recent report studying HPV-16 E6 memory T helper cells in healthy populations showed that the majority of responders targeted E6 residues (Welters et al., 2003). These data may suggest that amino acid changes in these regions may aid in escape of immune surveillance or render variants more susceptible to HIV-induced immunosuppression. However, in our study no significant increases were observed in the prevalence of either Asian American variants or pooled nt 350 G variants with decreasing CD4 cell counts (<200 cells mm 23 ). This lack of significance could have arisen owing to restrictive sample sizes; indeed, post hoc power calculations revealed a power of 17 % in detecting a significant difference in prevalence of variants with the nt 350 G mutation compared with the European 350T prototype across CD4 cell count strata. Several issues need to be addressed regarding our study. The observed significant associations were based on small sample sizes and these results need to be further validated in larger prospective studies. All the HIV-negative women had a previous history of cytological abnormalities and were recruited from colposcopy clinics. This high-risk nature of the control population may in fact have led to the lack of significant associations between variant lineages and cytological status. Our analyses combining all variants with a G-350 mutation included both European and non- European variants. The decision to compare the prevalence of any variant lineage harbouring an nt 350 TRG mutation was made a priori and was based on the use of similar comparisons/grouping in previous studies (Andersson et al., 2000; Zehbe et al., 2001; Londesborough et al., 1996). This grouping may be inappropriate considering the biological differences between European and non-european variant lineages. In conclusion, we characterized the prevalence of HPV-16 E6 variant lineages in HIV-positive and -negative women. We report an increase in the prevalence of Asian American variants and variants with an nt 350 G mutation in HIVpositive women. Studies are required to elucidate further the influence of immunosuppression on variant infections. ACKNOWLEDGEMENTS Financial support: Doris Duke clinical research grant, National Cancer Institute (grant NCI-1 R03 CA86378) and Health Excellency Fund of Louisiana. REFERENCES Ahdieh, L., Klein, R. S., Burk, R. & 7 other authors (2001). 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