Impact of age at vaccination and cervical HPV infection status on binding and neutralizing antibody titers at 10 years after receiving single or higher doses of quadrivalent HPV vaccine.

Long-term follow-up of a cohort of unmarried girls who received one, two, or three doses of quadrivalent HPV vaccine, between 10 and 18 years of age, in an Indian multi-centric study allowed us to compare antibody responses between the younger and older age cohorts at 10-years post-vaccination, and study the impact of initiation of sexual activity and cervical HPV infections on antibody levels. Among the younger (10-14 years) recipients of a single dose, 97.7% and 98.2% had detectable binding antibody titers against HPV 16 and HPV 18 respectively at ten years post-vaccination. The proportions among those receiving a single dose at age 15-18 years were 92.3% and 94.2% against HPV 16 and HPV 18 respectively. Mean HPV 16 binding antibody titers were 2.1 folds (95%CI 1.4 to 3.3) higher in those vaccinated at ages 10-14 years, and 1.9 folds (95%CI 1.2 to 3.0) higher in those vaccinated at 15-18 years compared to mean titers seen in the unvaccinated women. Compared to previous timepoints of 36 or 48 months, binding antibodies against HPV 16 and neutralizing antibodies against both HPV 16 and HPV 18 were significantly higher at 10 years. This rise was more pronounced in participants vaccinated at 15-18 years. No association of marital status or cervical HPV infections was observed with the rise in titer. Durability of antibody response in single dose recipients correlated well with the high efficacy of a single dose against persistent HPV 16/18 infections irrespective of age at vaccination, as we reported earlier.

Development and Evaluation of p16 based Double Antibody Sandwich ELISA for Detection of Cervical Precancer and Cancer.

OBJECTIVE: Cervical cancer is the third most common cancer in women, worldwide. This study was designed to develop an affordable, accurate and simpler screening test like Enzyme-linked immunosorbent assay (ELISA) which is low cost and will help in bringing down the disease burden in resource poor countries. METHODS: In this study, we have raised and evaluated monoclonal antibodies against recombinant p16 using immunohistochemistry (IHC), western blot, immunoprecipitation and ELISA. Double antibody sandwich ELISA (DAS-ELISA) and cytokeratin ELISA was designed for screening women with cervical dysplasia and cancer. RESULTS: Cloned, expressed and purified recombinant p16 were used for generation of monoclonal antibodies. After initial screening, six clones were selected, and affinity purified. Except 155D11G10, which was isotype Immunoglobulin (Ig) G1 all the others were found to be IgG2b. 133A6G5 and 151A7B9 were found to be best for p16 IHC, both showed 70 - 80% and 80 - 90% of nuclear staining respectively. All the antibodies positively detected p16 from the HeLa lysates in western blot except 133A6G5. Studies using immunoprecipitation showed 133A6G5, specifically detected p16. DAS-ELISA developed using a combination of our p16 monoclonal antibodies showed sensitivity of up to 2pg. A pilot study using DAS-ELISA and cytokeratin ELISA in cervical samples revealed the assay sensitivity and specificity as 100% and 80%, respectively. CONCLUSION: Using combination of DAS-ELISA and cytokeratin ELISA we have developed an accurate and reliable method for the early detection of cervical cancer in a subject, with minimal false results. In the future after large scale validation, p16 ELISA could be used as a reliable tool for diagnostic purposes.

Evaluation of immune response to single dose of quadrivalent HPV vaccine at 10-year post-vaccination.

BACKGROUND: The recent World Health Organization recommendation supporting single-dose of HPV vaccine will significantly reduce programmatic cost, mitigate the supply shortage, and simplify logistics, thus allowing more low- and middle-income countries to introduce the vaccine. From a programmatic perspective the durability of protection offered by a single-dose will be a key consideration. The primary objectives of the present study were to determine whether recipients of a single-dose of quadrivalent HPV vaccine had sustained immune response against targeted HPV types (HPV 6,11,16,18) at 10 years post-vaccination and whether this response was superior to the natural antibody titres observed in unvaccinated women. METHODS: Participants received at age 10-18 years either one, two or three doses of the quadrivalent HPV vaccine. Serology samples were obtained at different timepoints up to 10 years after vaccination from a convenience sample of vaccinated participants and from age-matched unvaccinated women at one timepoint. The evolution of the binding and neutralizing antibody response was presented by dose received. 10-year durability of immune responses induced by a single-dose was compared to that after three doses of the vaccine and in unvaccinated married women. RESULTS: The dynamics of antibody response among the single-dose recipients observed over 120 months show stabilized levels 18 months after vaccination for all four HPV types. Although the HPV type-specific (binding or neutralizing) antibody titres after a single-dose were significantly inferior to those after three doses of the vaccine (lower bounds of GMT ratios < 0.5), they were all significantly higher than those observed in unvaccinated women following natural infections (GMT ratios: 2.05 to 4.04-fold higher). The results correlate well with the high vaccine efficacy of single-dose against persistent HPV 16/18 infections reported by us earlier at 10-years post-vaccination. CONCLUSION: Our study demonstrates the high and durable immune response in single-dose recipients of HPV vaccine at 10-years post vaccination.

Vaccine efficacy against persistent human papillomavirus (HPV) 16/18 infection at 10 years after one, two, and three doses of quadrivalent HPV vaccine in girls in India: a multicentre, prospective, cohort study.

BACKGROUND: A randomised trial designed to compare three and two doses of quadrivalent human papillomavirus (HPV) vaccine in adolescent girls in India was converted to a cohort study after suspension of HPV vaccination in trials by the Indian Government. In this Article, the revised aim of the cohort study was to compare vaccine efficacy of single dose to that of three and two doses in protecting against persistent HPV 16 and 18 infection at 10 years post vaccination. METHODS: In the randomised trial, unmarried girls aged 10-18 years were recruited from nine centres across India and randomly assigned to either two doses or three doses of the quadrivalent HPV vaccine (Gardasil [Merck Sharp & Dohme, Whitehouse Station, NJ, USA]; 0·5 mL administered intramuscularly). After suspension of recruitment and vaccination, the study became a longitudinal, prospective cohort study by default, and participants were allocated to four cohorts on the basis of the number vaccine doses received per protocol: the two-dose cohort (received vaccine on days 1 and 180 or later), three-dose cohort (days 1, 60, and 180 or later), two-dose default cohort (days 1 and 60 or later), and the single-dose default cohort. Participants were followed up yearly. Cervical specimens were collected from participants 18 months after marriage or 6 months after first childbirth, whichever was earlier, to assess incident and persistent HPV infections. Married participants were screened for cervical cancer as they reached 25 years of age. Unvaccinated women age-matched to the married vaccinated participants were recruited to serve as controls. Vaccine efficacy against persistent HPV 16 and 18 infections (the primary endpoint) was analysed for single-dose recipients and compared with that in two-dose and three-dose recipients after adjusting for imbalance in the distribution of potential confounders between the unvaccinated and vaccinated cohorts. This trial is registered with ISRCTN, ISRCTN98283094, and ClinicalTrials.gov, NCT00923702. FINDINGS: Vaccinated participants were recruited between Sept 1, 2009, and April 8, 2010 (date of vaccination suspension), and followed up over a median duration of 9·0 years (IQR 8·2-9·6). 4348 participants had three doses, 4980 had two doses (0 and 6 months), and 4949 had a single dose. Vaccine efficacy against persistent HPV 16 and 18 infection among participants evaluable for the endpoint was 95·4% (95% CI 85·0-99·9) in the single-dose default cohort (2135 women assessed), 93·1% (77·3-99·8) in the two-dose cohort (1452 women assessed), and 93·3% (77·5-99·7) in three-dose recipients (1460 women assessed). INTERPRETATION: A single dose of HPV vaccine provides similar protection against persistent infection from HPV 16 and 18, the genotypes responsible for nearly 70% of cervical cancers, to that provided by two or three doses. FUNDING: Bill & Melinda Gates Foundation.

Acquisition, prevalence and clearance of type-specific human papillomavirus infections in young sexually active Indian women: A community-based multicentric cohort study.

In context of the ongoing multi-centric HPV vaccine study in India, unvaccinated married women (N = 1484) aged 18-23 years were recruited in 2012-2015 as age-matched controls to the vaccinated women and followed up yearly. We assess type-specific prevalence, natural history and potential determinants of human papillomavirus (HPV) infection in these unvaccinated women. Cervical samples were collected yearly for at least four consecutive years. A Multiplex Type-Specific E7-Based polymerase chain reaction assay was used to detect 21 HPV types. HPV prevalence was 36.4% during 6 years. Most common HPV types were 16 (6.5%) and 31 (6.1%). Highest persistence were observed for HPV 35 (62.5%) and 52 (25%). New HPV acquisition rate was 5.6/1000 person-months of observation (PMO), highest for HPV 16 (1.1/1000 PMO). Type-specific clearance rates ranged between 2.9-5.5/100 PMO. HPV 16 and/or 18 infections were 41% (95% CI 4-63%) lower among women with 2-<3 years between marriage and first cervical sample collection compared to those with <2 years. HPV prevalence and acquisition rates in young Indian women were lower than their Western counterparts. HPV 16 infections being most common shows the importance and potential impact of HPV vaccination in India. Women with 2-3 years exposure had reduced risk possibly due to higher infections clearance.

Accuracy of concurrent visual and cytology screening in detecting cervical cancer precursors in rural India.

The high burden of cervical cancer and inadequate/suboptimal cytology screening in developing countries led to the evaluation of visual screening tests, like visual inspection with acetic acid (VIA) and Lugol's iodine (VILI). We describe the performance of VIA, VILI and cytology, carried out in a multinational project called "Screening Technologies to Advance Rapid Testing" in 5,519 women aged 30-49 years, in detecting cervical intraepithelial neoplasia (CIN). VIA, VILI and cytology were positive in 16.9%, 15.6% and 6.1% women, respectively. We found 57 cases of CIN2, 55 of CIN3 and 12 of cervical cancer; 90% of CIN3 and 43% CIN2 cases were positive for p16 overexpression and high-risk HPV infection, indicating a high validity of histological diagnosis. The sensitivity of VIA, VILI and cytology to detect high-grade CIN were 64.5%, 64.5% and 67.7%, respectively; specificities were 84.2%, 85.5% and 95.4%. A high proportion of p16 positive CIN 3 (93.8%) and 2 (76.9%) were positive on cytology compared with visual tests (68.8% and 53.8%, respectively) indicating a higher sensitivity of cytology to detect p16 positive high-grade CIN. However, the immediate availability of the results from the visual tests permits diagnosis and/or treatment to be performed in the same sitting, which can potentially reduce loss to follow-up when women must be recalled following positive cytology. Organizing visual screening services in low-resource countries may facilitate the gradual building of an infrastructure committed to screening allowing the eventual introduction of more sensitive, highly objective, reproducible and affordable human papillomavirus screening tests in future.