Prevalent high-risk HPV infection and vaginal microbiota in Nigerian women.

In this study, we evaluated the association between high-risk human papillomavirus (hrHPV) and the vaginal microbiome. Participants were recruited in Nigeria between April and August 2012. Vaginal bacterial composition was characterized by deep sequencing of barcoded 16S rRNA gene fragments (V4) on Illumina MiSeq and HPV was identified using the Roche Linear Array® HPV genotyping test. We used exact logistic regression models to evaluate the association between community state types (CSTs) of vaginal microbiota and hrHPV infection, weighted UniFrac distances to compare the vaginal microbiota of individuals with prevalent hrHPV to those without prevalent hrHPV infection, and the Linear Discriminant Analysis effect size (LEfSe) algorithm to characterize bacteria associated with prevalent hrHPV infection. We observed four CSTs: CST IV-B with a low relative abundance of Lactobacillus spp. in 50% of participants; CST III (dominated by L. iners) in 39·2%; CST I (dominated by L. crispatus) in 7·9%; and CST VI (dominated by proteobacteria) in 2·9% of participants. LEfSe analysis suggested an association between prevalent hrHPV infection and a decreased abundance of Lactobacillus sp. with increased abundance of anaerobes particularly of the genera Prevotella and Leptotrichia in HIV-negative women (P < 0·05). These results are hypothesis generating and further studies are required.

Immune responses elicited by a fourth dose of the HPV-16/18 AS04-adjuvanted vaccine in previously vaccinated adult women.

BACKGROUND: Vaccines are now available for the prevention of HPV-16/18-related cervical infections and pre-cancers, primarily targeting adolescent girls. Since the risk of HPV exposure potentially persists throughout a woman's sexual life, vaccine-derived immunity should be long-term. The current study, HPV-024 (NCT00546078, http://clinicaltrials.gov), assessed the immune memory in North American women who received three doses of HPV-16/18 AS04-adjuvanted vaccine 7 years earlier in HPV-001 (NCT00689741). METHODS: Women vaccinated in HPV-001 received a 4th-dose of the HPV-16/18 vaccine (024-4DV group, N=65). Post 4th-dose immune responses were compared with post 1st-dose immune responses in cross-vaccination controls (024-3DV group, N=50). Reactogenicity was compared between the 4th-dose and the 1st-dose administration. RESULTS: Pre 4th-dose, 100% of subjects in the 024-4DV group remained seropositive for anti-HPV-16/18 antibodies (ELISA). Compared to pre 4th-dose, GMTs for anti-HPV-16 and anti-HPV-18 antibodies were respectively 9.3-fold and 8.7-fold higher at day 7, and 22.7-fold and 17.2-fold higher at month 1. Compared to post 1st-dose, GMTs for anti-HPV-16 and anti-HPV-18 were respectively 80.5-fold and 205.4-fold higher at day 7, and 11.8-fold and 20.5-fold higher at month 1. Furthermore, 68.2% and 77.3% of women had HPV-16/18 specific memory B-cells, respectively, pre 4th-dose, rising to 100% one month post 4th-dose vaccination. The 4th-dose was generally well tolerated. CONCLUSION: A 4th-dose of HPV-16/18 AS04-adjuvanted vaccine triggered a rapid and strong anamnestic response in previously vaccinated women, demonstrating vaccine-induced immune memory.

Efficacy of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine in women aged 15-25 years with and without serological evidence of previous exposure to HPV-16/18.

In the Phase III PATRICIA study (NCT00122681), the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine (Cervarix(®), GlaxoSmithKline Biologicals) was highly efficacious against HPV-16/18 infections and precancerous lesions in women HPV-16/18 deoxyribose nucleic acid (DNA) negative and seronegative at baseline. We present further data on vaccine efficacy (VE) against HPV-16/18 in the total vaccinated cohort including women who may have been exposed to HPV-16/18 infection before vaccination. In women with no evidence of current or previous HPV-16/18 infection (DNA negative and seronegative), VE was 90.3% (96.1% confidence interval: 87.3-92.6) against 6-month persistent infection (PI), 91.9% (84.6-96.2) against cervical intraepithelial neoplasia (CIN)1+ and 94.6% (86.3-98.4) against CIN2+ [97.7% (91.1-99.8) when using the HPV type assignment algorithm (TAA)]. In women HPV-16/18 DNA negative but with serological evidence of previous HPV-16/18 infection (seropositive), VE was 72.3% (53.0-84.5) against 6-month PI, 67.2% (10.9-89.9) against CIN1+, and 68.8% (-28.3-95.0) against CIN2+ [88.5% (10.8-99.8) when using TAA]. In women with no evidence of current HPV-16/18 infection (DNA negative), regardless of their baseline HPV-16/18 serological status, VE was 88.7% (85.7-91.1) against 6-month PI, 89.1% (81.6-94.0) against CIN1+ and 92.4% (84.0-97.0) against CIN2+ [97.0% (90.6-99.5) when using TAA]. In women who were DNA positive for one vaccine type, the vaccine was efficacious against the other vaccine type. The vaccine did not impact the outcome of HPV-16/18 infections present at the time of vaccination. Vaccination was generally well tolerated regardless of the woman's HPV-16/18 DNA or serological status at entry.

Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years.

BACKGROUND: Prophylactic human papillomavirus (HPV) vaccines have to provide sustained protection. We assessed efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. METHODS: Women aged 15-25 years, with normal cervical cytology, who were HPV-16/18 seronegative and oncogenic HPV DNA-negative (14 types) at screening participated in a double-blind, randomised, placebo-controlled initial study (n=1113; 560 vaccine group vs 553 placebo group) and follow-up study (n=776; 393 vs 383). 27 sites in three countries participated in the follow-up study. Cervical samples were tested every 6 months for HPV DNA. Management of abnormal cytologies was prespecified, and HPV-16/18 antibody titres were assessed. The primary objective was to assess long-term vaccine efficacy in the prevention of incident cervical infection with HPV 16 or HPV 18, or both. We report the analyses up to 6.4 years of this follow-up study and combined with the initial study. For the primary endpoint, the efficacy analysis was done in the according-to-protocol (ATP) cohort; the analysis of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was done in the total vaccinated cohort (TVC). The study is registered with ClinicalTrials.gov, number NCT00120848. FINDINGS: For the combined analysis of the initial and follow-up studies, the ATP efficacy cohort included 465 women in the vaccine group and 454 in the placebo group; the TVC included 560 women in the vaccine group and 553 in the placebo group. Vaccine efficacy against incident infection with HPV 16/18 was 95.3% (95% CI 87.4-98.7) and against 12-month persistent infection was 100% (81.8-100). Vaccine efficacy against CIN2+ was 100% (51.3-100) for lesions associated with HPV-16/18 and 71.9% (20.6-91.9) for lesions independent of HPV DNA. Antibody concentrations by ELISA remained 12-fold or more higher than after natural infection (both antigens). Safety outcomes were similar between groups: during the follow-up study, 30 (8%) participants reported a serious adverse event in the vaccine group versus 37 (10%) in the placebo group. None was judged related or possibly related to vaccination, and no deaths occurred. INTERPRETATION: Our findings show excellent long-term efficacy, high and sustained immunogenicity, and favourable safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. FUNDING: GlaxoSmithKline Biologicals (Belgium).

Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women.

BACKGROUND: The human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine was immunogenic, generally well tolerated, and effective against HPV-16 or HPV-18 infections, and associated precancerous lesions in an event-triggered interim analysis of the phase III randomised, double-blind, controlled PApilloma TRIal against Cancer In young Adults (PATRICIA). We now assess the vaccine efficacy in the final event-driven analysis. METHODS: Women (15-25 years) were vaccinated at months 0, 1, and 6. Analyses were done in the according-to-protocol cohort for efficacy (ATP-E; vaccine, n=8093; control, n=8069), total vaccinated cohort (TVC, included all women receiving at least one vaccine dose, regardless of their baseline HPV status; represents the general population, including those who are sexually active; vaccine, n=9319; control, n=9325), and TVC-naive (no evidence of oncogenic HPV infection at baseline; represents women before sexual debut; vaccine, n=5822; control, n=5819). The primary endpoint was to assess vaccine efficacy against cervical intraepithelial neoplasia 2+ (CIN2+) that was associated with HPV-16 or HPV-18 in women who were seronegative at baseline, and DNA negative at baseline and month 6 for the corresponding type (ATP-E). This trial is registered with ClinicalTrials.gov, number NCT00122681. FINDINGS: Mean follow-up was 34.9 months (SD 6.4) after the third dose. Vaccine efficacy against CIN2+ associated with HPV-16/18 was 92.9% (96.1% CI 79.9-98.3) in the primary analysis and 98.1% (88.4-100) in an analysis in which probable causality to HPV type was assigned in lesions infected with multiple oncogenic types (ATP-E cohort). Vaccine efficacy against CIN2+ irrespective of HPV DNA in lesions was 30.4% (16.4-42.1) in the TVC and 70.2% (54.7-80.9) in the TVC-naive. Corresponding values against CIN3+ were 33.4% (9.1-51.5) in the TVC and 87.0% (54.9-97.7) in the TVC-naive. Vaccine efficacy against CIN2+ associated with 12 non-vaccine oncogenic types was 54.0% (34.0-68.4; ATP-E). Individual cross-protection against CIN2+ associated with HPV-31, HPV-33, and HPV-45 was seen in the TVC. INTERPRETATION: The HPV-16/18 AS04-adjuvanted vaccine showed high efficacy against CIN2+ associated with HPV-16/18 and non-vaccine oncogenic HPV types and substantial overall effect in cohorts that are relevant to universal mass vaccination and catch-up programmes. FUNDING: GlaxoSmithKline Biologicals.

Serum carotenoids and risk of cervical intraepithelial neoplasia in Southwestern American Indian women.

The objective of this research was to evaluate the association between serum carotenoids and cervical intraepithelial neoplasia (CIN) among Southwestern American Indian women. Cases were American Indian women with biopsy-proven CIN II/III cervical lesions (n = 81) diagnosed between November 1994 and October 1997. Controls were American Indian women from the same clinics with normal cervical epithelium (n = 160). All of the subjects underwent interviews and laboratory evaluations. Interviews evaluated demographic information, sexual history, and cigarette smoking. Serum concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, and lutein/zeaxanthin were measured by high performance liquid chromatography. Cervical human papillomavirus infection was detected using a PCR-based test. Increasing levels of alpha-carotene, beta-cryptoxanthin, and lutein/zeaxanthin were associated with decreasing risk of CIN II/III. In addition, the highest tertiles of beta-cryptoxanthin (odds ratio = 0.39, 95% confidence interval = 0.17-0.91) and lutein/zeaxanthin (odds ratio = 0.40, 95% confidence interval = 0.17-0.95) were associated with the lowest risk of CIN. In conclusion, specially targeted intervention efforts to increase consumption of fruits and vegetables may protect Southwestern American Indian women from developing CIN.

Human leukocyte antigen class I and II alleles and risk of cervical neoplasia: results from a population-based study in Costa Rica.

To examine human leukocyte antigen (HLA) involvement in the development of all grades of cervical neoplasia, a nested case-control study of 10,077 women in Guanacaste, Costa Rica, was conducted. Participants had invasive cervical cancer, high-grade squamous intraepithelial lesions (HSILs; n=166), or low-grade squamous intraepithelial lesions (LSILs); were positive for human papillomavirus (HPV) with no evidence of cervical neoplasia (n=320); or were HPV negative with no evidence of cervical neoplasia but with a history of high-risk sexual behavior (n=173). Compared with women who were HPV negative, women with HLA-DRB1*1301 were associated with decreased risk for cancer/HSILs (odds ratio [OR], 0.4; 95% confidence interval [CI], 0.2-0.7) and for LSILs/HPV (OR, 0.6; 95% CI, 0.3-0.9). Women with both HLA-B*07 and HLA-DQB1*0302 had an 8.2-fold increased risk for cancer/HSILs (95% CI, 1.8-37.2) and a 5.3-fold increased risk for LSILs/HPV (95% CI, 1.2-23.7). These results support the hypothesis that multiple risk alleles are needed in order to increase risk for cervical neoplasia, but a single protective allele may be sufficient for protection.

Neutralization of human papillomavirus type 11 (HPV-11) by serum from women vaccinated with yeast-derived HPV-11 L1 virus-like particles: correlation with competitive radioimmunoassay titer.

Neutralization of human papillomavirus type 11 (HPV-11) has been demonstrated using serum and cervical secretions from primates vaccinated with virus-like particles (VLPs). Theoretically, neutralizing antibodies could protect women from HPV infection. The immunogenicity of a yeast-derived HPV-11 L1 VLP vaccine was tested in women. Serum specimens were evaluated for HPV-11 titer by competitive radioimmunoassay (cRIA) and for neutralization by use of the athymic mouse xenograft system. Analysis of serum from 104 subjects showed a dose response in HPV-11 cRIA titers and neutralization. Overall, 68 (82.9%) of 82 postimmunization serum specimens from VLP recipients were 100% neutralizing when used in the assay at a 1:50 dilution. Of 69 serum specimens, 63 (91.3%) with cRIA titers >200 milliMerck units per milliliter were neutralizing. Immunization with HPV VLPs elicits a vigorous serum immune response in a high percentage of women. The HPV-11 cRIA titer appears to be a surrogate marker for neutralization.

Determinants of genital human papillomavirus detection in a US population.

This study investigated the association of selected demographic and behavioral characteristics with the detection of low-risk, high-risk, and uncharacterized genital human papillomavirus (HPV) in women attending clinic for routine nonreferral gynecologic health care. Cervical specimens obtained from 3863 women 18-40 years old (mean, 28 years) with no history of high-grade cervical disease were analyzed for 38 HPV types. Overall, HPV prevalence was 39.2%. The prevalence of high-risk, low-risk, and uncharacterized HPV types was 26.7%, 14.7%, and 13.0%, respectively. As expected, the characteristics most strongly associated with overall HPV detection were age and numbers of lifetime and recent sex partners. Low-risk, high-risk, and uncharacterized HPV detection increased with increasing numbers of sex partners. There was a decline in high-risk and low-risk HPV detection with increasing age but little change in uncharacterized HPV detection. These results suggest that the uncharacterized HPV types have a different natural history than either low-risk or high-risk HPV types.

Human papillomavirus: a highly prevalent sexually transmitted disease agent among female sex workers from Mexico City.

BACKGROUND: Cervical cancer, a human papillomavirus (HPV)-caused neoplasia, is highly prevalent in Mexico. GOAL: To determine the prevalence of HPV infection in female sex workers (FSW) from Mexico City and to assess the association between HPV infection and the characteristics of these women. STUDY DESIGN: A questionnaire was applied to 495 FSW. Cervical cell specimens were obtained for DNA amplification and hybridization to detect 27 HPV types. A risk factor analysis was performed. RESULTS: The overall prevalence of HPV infection was 48.9%. The prevalence of high-risk HPV types was 43%, whereas that of low-risk types was 24.6%. A total of 18.8% of study participants was infected with both high-risk and low-risk HPV types, and 28.5% were infected with two or more HPV types. Younger age and failure to use a condom were independently associated HPV risks (odds ratio, 7.3 and 2.3; 95% CI, 3.5-15.0 and 1.2-4.4, respectively). CONCLUSIONS: Infection with high-risk and multiple HPV types is high among Mexican FSW. This study corroborated a higher infection rate in younger women. A higher risk of HPV infection is also observed in women who have been involved with sex work for less than 1 year. However, condom use showed a protective effect against HPV infection.

Contraceptive and reproductive risk factors for cervical intraepithelial neoplasia in American Indian women.

OBJECTIVES: To evaluate contraceptive and reproductive risk factors for cervical intraepithelial neoplasia (CIN) in southwestern American Indian women. METHODS: We conducted a clinic-based case-control study. Cases were American Indian women with biopsy-proven CIN I, CIN II or CIN III. Controls were from the same clinics and had normal cervical epithelium. All subjects underwent structured interviews focused on contraceptive and reproductive factors. Laboratory assays included polymerase chain reaction (PCR)-based tests for cervical human papillomavirus (HPV) infection. RESULTS: We enrolled 628 women in the study. The strongest risk factors for CIN II/III included HPV infection (adjusted odds ratio [OR] = 7.9, 95% CI : 4.7-13.2), and low income (OR = 3.1, 95% CI : 1.7-5.7). The use of an intrauterine device (IUD) ever (OR = 3.0, 95% CI : 1.4-6.1) and currently (OR = 4.1, 95% CI : 1.1-14.6), and > or = 3 vaginal deliveries (OR = 5.2, 95% CI : 2.4-11.1) were associated with CIN II/III. History of infertility was also associated with CIN II/III (OR = 2.1, 95% CI : 1.0-4.2). CONCLUSIONS: The data suggest that history of infertility, IUD use and vaginal deliveries were associated with CIN among American Indian women.

Risk factors for cervical intraepithelial neoplasia in southwestern American Indian women.

The authors assessed risk factors for cervical intraepithelial neoplasia (CIN) among southwestern American Indian women using case-control methods. Cases were New Mexico American Indian women with biopsy-proven grade I (n = 190), grade II (n = 70), or grade III (n = 42) cervical lesions diagnosed between November 1994 and October 1997. Controls were American Indian women from the same Indian Health Service clinics with normal cervical epithelium (n = 326). All subjects underwent interviews and laboratory evaluations. Interviews focused on history of sexually transmitted diseases, sexual behavior, and cigarette smoking. Laboratory assays included polymerase chain reaction-based tests for cervical human papillomavirus infection, tests for gonorrhea and chlamydia, wet mounts, and serologic assays for antibodies to Treponema pallidum, herpes simplex virus, and hepatitis B and C viruses. In multiple logistic regression analysis, the strongest risk factors for CIN II/III among American Indian women were human papillomavirus type 16 infection (adjusted odds ratio (OR) = 7.6; 95% confidence interval (CI): 2.4, 23.2), any human papillomavirus infection (OR = 5.8; 95% CI: 3.3, 10.0), low income (OR = 3.3; 95% CI: 1.7, 6.2), and history of any sexually transmitted disease (OR = 2.0; 95% CI: 1.1, 3.5). Unlike previous research, this study found no strong associations between CIN and sexual activity or cigarette smoking.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

Comparison of variant-specific hybridization and single-strand conformational polymorphism methods for detection of mixed human papillomavirus type 16 variant infections.

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.

Risk factors for rapid-onset cervical cancer.

OBJECTIVES: The current study was designed to elucidate risk factors associated with the development of cervical cancer during the course of routine Papanicolaou smear screening (rapid-onset cervical cancer). STUDY DESIGN: Four hundred eighty-three women diagnosed with invasive cervical cancer, representing 73% of all such tumors diagnosed in Connecticut between 1985 and 1990, were studied. Papanicolaou smear screening and risk factor information was obtained by questionnaire and physician record review. Results from human papillomavirus deoxyribonucleic acid testing by polymerase chain reaction of tumor samples were available for 278 study participants. Prediagnostic Papanicolaou smear slides were reviewed for 67% of cases with a screening history. Screening history information, slide review, and questionnaire data were used to classify women as having rapid-onset cervical cancer (n = 43), possible rapid-onset cervical cancer (n = 111), or normal-onset cervical cancer (n = 329). RESULTS: Compared with normal-onset cases, rapid-onset cases tended to be younger (P =.001) and were more likely to be white (P =.002), diagnosed with adenocarcinomas or adenosquamous carcinomas (P =.001), and diagnosed with early-stage disease (P =.001). Cases diagnosed as possible rapid-onset disease tended to have a profile that was intermediate to that observed for rapid-onset and normal-onset cases. Human papillomavirus deoxyribonucleic acid was detected in 75.2% of cases tested. Compared with women who tested positive for human papillomavirus type 16 or other, those positive for human papillomavirus type 18 had a relative risk for rapid-onset disease of 1.6 (95% confidence interval 0.52-4.9). No significant association was observed between type 18 and possible rapid-onset disease when possible rapid-onset cases were compared with women diagnosed with normal-onset cervical cancer (relative risk 0.67, 95% confidence interval 0.29-1.6). Oral contraceptive use, cigarette smoking, number of pregnancies, and a maternal history of cervical cancer were not significantly associated with rapid-onset disease. CONCLUSIONS: Results from this study suggest that the risk factors associated with the development of rapid-onset cervical cancer are similar to those for normal-onset disease.

Human papillomavirus type 16 variant lineages characterized by nucleotide sequence analysis of the E5 coding segment and the E2 hinge region.

We have previously examined 29 cervical cell isolates for human papillomavirus type 16 (HPV-16) sequence variations in the E6, L2 and L1 coding regions, and the long control region (LCR). Twenty-five of these isolates as well as 23 additional isolates are characterized here as we present the complete E5 coding segment and the E2 hinge region. Eight amino acid variations were observed in the E5 coding segment, 13 were identified in the E2 hinge region and 5 were observed in the overlapping E4 coding segment. These amino acid variations may be relevant to differences in biological functions and may result in altered humoral or cell-mediated immune responses to HPV-16 variants. The characterization of sequence variation within high-risk HPV types might be important in the search for epidemiological correlates of cervical cancer risk. This work complements and extends HPV-16 genome sequence information from specific isolates previously reported by our group.

Comparison of PCR- and hybrid capture-based human papillomavirus detection systems using multiple cervical specimen collection strategies.

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.

Risk of anal carcinoma in situ in relation to human papillomavirus type 16 variants.

Infection with human papillomavirus (HPV), especially HPV16, is central to the development of squamous anogenital cancers and their precursor lesions, termed "squamous intraepithelial neoplasias." Men who have sex with men, particularly those who are infected with HIV, are at a high risk for anal infection with HPV16 and for low-grade anal neoplasia; however, only a subset of these men develop anal invasive cancer or its immediate precursor lesion, anal carcinoma in situ (CIS). To examine the hypothesis that certain variants of HPV16 are most strongly associated with development of anal CIS, we followed 589 men who have sex with men whose initial anal cytological smears did not show anal CIS. Anoscopy, anal cytology, and PCR-based assays for detection and classification of HPV types were performed every 4-6 months, with HPV16 further classified by single-stranded conformation polymorphism analysis as being a prototype-like (PL) or non-prototype-like (NPL) variant. Anal CIS was histologically confirmed in 6 of 384 (1.6%) consistently HPV16-negative men, in 12 of 183 (6.6%) men with HPV16 PL variants, and in 4 of 22 (18.2%) men with HPV16 NPL variants. After adjustment for anal cytological diagnoses at study entry, HIV status and CD4 count, and detection of HPV types other than type 16, men with HPV16 NPL variants were 3.2 times (95% confidence interval, 1.0-10.3) more likely to develop anal CIS than were those with PL variants. Neither detection of HPV16 DNA at high levels nor detection of HPV16 DNA for a prolonged period, factors that we previously demonstrated to be associated with risk of high-grade anal squamous intraepithelial neoplasia, was significantly associated with HPV16 NPL variants. The biological mechanism relating to Ihis excess risk remains undetermined.

Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

Preventive vaccines for cervical cancer.

The potential use of vaccines for the human papillomavirus (HPV) in the prevention and treatment of cervical cancer is a possibility in the near future. Close to 20 genotypes of HPV, of the 75 that have been identified, infect the feminine genital tract, but four subtypes (16, 18, 31 and 45) have been associated in close to 80% of cervical cancers, this article proposes that in order to design an effective prophylactic vaccine against HPV infection, an adequate immune response should be guaranteed through four goals; a) activation of antigens present in the cell; b) overcoming the host response and viral genetic variability in the T cell response; c) generation of high levels of T and B memory cells; and d) persistence of antigens.