John Schussler

Human papillomavirus variants are classified into lineages based on their whole genome sequence, but the impact of lineage variation on the structure or function of encoded proteins is unclear. We used a global panel of lineage-specific natural infection sera to assess antibody-binding specificity for lineage-specific L1L2 antigens, and we used these data to create relational antigenic maps. Merging these data with neutralizing antibody data demonstrated similar spatial geometry and revealed dependency on a limited number of amino residues on the capsid surface. These data inform the degree of lineage specificity within the natural infection humoral immune response to oncogenic human papillomaviruses.

Background: Cervical cancer is caused by persistent infection with carcinogenic human papillomavirus (HPV) genotypes. Prophylactic HPV vaccines are highly efficacious in preventing the acquisition of HPV infection. HPV vaccine trials and epidemiologic studies based on virologic endpoints rely on valid and reproducible measurements of HPV. We evaluated the second version of TypeSeq (TS2), a next-generation, sequencing-based assay that detects 46 HPV genotypes, in a historical phase 3 clinical trial. Methods: We used 1214 stored cervical samples from women enrolled in the Costa Rica HPV Vaccine Trial with available HPV results from Short PCR Fragment 10- Line Probe Assay 25 (SPF10-LiPA25). TS2 was first validated at the National Cancer Institute (NCI) and transferred to the laboratory in Costa Rica, where we conducted a second validation study. We compared TS2 results generated at each laboratory to the SPF10-LiPA25 results. Results: Overall, each laboratory demonstrated high positive agreement for most carcinogenic and noncarcinogenic genotypes between TS2 and SPF10-LiPA25. Intralaboratory comparisons revealed very high agreement in repeated testing. Interlaboratory comparisons showed high agreement for most carcinogenic and noncarcinogenic types. Overall, there were no statistically significant differences in vaccine efficacy in the according-to-protocol cohort using TS2 (either in NCI or Costa Rica) or SPF10-LiPA25 (McNemar P values >.05). Costa Rica produced similar vaccine efficacy estimates as NCI for HPV16/18, HPV31/33/45, and HPV35/39/51/52/56/58/59 as NCI (P values ≥.36). Conclusions: Compared to SPF10-LiPA25, a well-established standard for HPV genotyping, TS2 demonstrated high accuracy. Inter- and intralaboratory comparisons demonstrated that TS2 is valid and reproducible. TS2 can accurately classify the presence of HPV, which is essential in HPV vaccine trials evaluating virological endpoints. Clinical Trials Registration: NCT00128661.

Background: Human papillomavirus (HPV) type 16 E6 protein seropositivity accurately predicts oropharyngeal squamous cell carcinoma risk decades before diagnosis; but the biomarker’s translational potential is unknown. To inform considerations for oropharyngeal squamous cell carcinoma screening, we described HPV-16 E6 seroprevalence, predictors, and kinetics among cancer-free men. Methods: In a cohort study in Brazil, Mexico, and the United States, we calculated HPV-16 E6 seropositivity (median fluorescence intensity > 1000), measured by multiplex serology, in cancer-free men. We assessed HPV-16 E6 seropositivity predictors using logistic regression, adjusting for country, age, sexual orientation, and lifetime number of sexual partners. Among HPV-16 E6 seropositive men, we retrieved all available retrospective serum samples and described temporal HPV-16 E6 antibody patterns. Results: Of 3997 men, 14 had HPV-16 E6 antibodies detected (seroprevalence = 0.35%, 95% CI = 0.19% to 0.59%; median fluorescence intensity = 2407 [IQR = 1325-5986]). Older age was associated with increased odds of HPV-16 E6 seropositivity (50-84 years vs 18-29 years odds ratio = 16.61, 95% CI = 2.20 to 417.03). Serum from 11 of the 14 seropositive men retested positive; 6 men had median fluorescence intensity above 5000, of whom 2 had median fluorescence intensity greater than 10 000. Seven men had 3 or more years of follow-up; all were persistently seropositive for 3 years. One man was seropositive for 9 years but seroreverted at his exit visit. Oral HPV-16 DNA (prevalence = 1.13%) was associated with HPV-16 E6 seropositivity (odds ratio = 16.87, 95% CI = 3.35 to 69.55), but oral HPV-16 DNA positivity was not persistent over follow-up, even when HPV-16 E6 antibodies were persistently detected. Conclusion: Although HPV-16 E6 seropositivity is rare, it is generally stable once detected; thus, HPV-16 E6 antibodies may be an informative biomarker of HPV-driven oropharyngeal squamous cell carcinoma. Few men seroreverted following HPV-16 E6 seropositivity but remained close to the seropositivity cutoff; thus, cancer risk among these men is less clear.

The AS04-adjuvanted human papillomavirus (HPV)16/18 vaccine, an L1-based vaccine, provides strong vaccine efficacy (VE) against vaccine-targeted type infections, and partial cross-protection to phylogenetically-related types, which may be affected by variant-level heterogeneity. We compared VE against incident HPV31, 33, 35, and 45 detections between lineages and SNPs in the L1 region among 2846 HPV-vaccinated and 5465 HPV-unvaccinated women through 11-years of follow-up in the Costa Rica HPV Vaccine Trial. VE was lower against HPV31-lineage-B (VE=60.7%;95%CI = 23.4%,82.8%) compared to HPV31-lineage-A (VE=94.3%;95%CI = 83.7%,100.0%) (VE-ratio = 0.64;95%CI = 0.25,0.90). Differential VE was observed at several lineage-associated HPV31-L1-SNPs, including a nonsynonymous substitution at position 6372 on the FG-loop, an important neutralization domain. For HPV35, the only SNP-level difference was at position 5939 on the DE-loop, with significant VE against nucleotide-G (VE=65.0%;95%CI = 28.0,87.8) but not for more the common nucleotide-A (VE=7.4%;95%CI = −34.1,36.7). Because of the known heterogeneity in precancer/cancer risk across cross-protected HPV genotype variants by race and region, our results of differential variant-level AS04-adjuvanted HPV16/18 vaccine efficacy has global health implications.

Human Papillomavirus (HPV) type variants have been classified into lineages and sublineages based upon their whole genome sequence. Here we have examined the specificity of antibodies generated following natural infection with lineage variants of oncogenic types (HPV16, 18, 31, 33, 45, 52 and 58) by testing serum samples assembled from existing archives from women residing in Africa, The Americas, Asia or Europe against representative lineage-specific pseudoviruses for each genotype. We have subjected the resulting neutralizing antibody data to antigenic clustering methods and created relational antigenic profiles for each genotype to inform the delineation of lineage-specific serotypes. For most genotypes, there was evidence of differential recognition of lineage-specific antigens and in some cases of a sufficient magnitude to suggest that some lineages should be considered antigenically distinct within their respective genotypes. These data provide compelling evidence for a degree of lineage specificity within the humoral immune response following natural infection with oncogenic HPV.

The HPV vaccine has shown sustained efficacy and consistent stabilization of antibody levels, even after a single dose. We defined the HPV16-VLP antibody avidity patterns over 11 years among women who received one- or three doses of the bivalent HPV vaccine in the Costa Rica HPV Vaccine Trial. Absolute HPV16 avidity was lower in women who received one compared to three doses, although the patterns were similar (increased in years 2 and 3 and remained stable over the remaining 8 years). HPV16 avidity among women who were HPV16-seropositive women at HPV vaccination, a marker of natural immune response to HPV16 infection, was significantly lower than those of HPV16-seronegative women, a difference that was more pronounced among one-dose recipients. No differences in HPV16 avidity were observed by HPV18 serostatus at vaccination, confirming the specificity of the findings. Importantly, point estimates for vaccine efficacy against incident, six-month persistent HPV16 infections was similar between women who were HPV16 seronegative and seropositive at the time of initial HPV vaccination for both one-dose and three-dose participants. It is therefore likely that this lower avidity level is still sufficient to enable antibody-mediated protection. It is encouraging for long-term HPV-vaccine protection that HPV16 antibody avidity was maintained for over a decade, even after a single dose.

Background: Previous reports from the Costa Rica Vaccine Trial demonstrated strong vaccine efficacy against HPV16/18 at the cervical, anal, and oral regions separately. However, the combined “woman-level” vaccine efficacy against infections at all three anatomic sites has not been examined in women with and without previous HPV16/18 exposure. Methods: Women aged 18-25 from the Costa Rica Vaccine Trial were randomized to be vaccinated with the HPV16/18 Vaccine (Cervarix) or a Hepatitis A vaccine at enrollment. Cervical samples were collected at every annual visit, while oral and anal samples were collected only at the four year follow-up visit. Samples were tested for alpha mucosal HPV DNA types utilizing the SPF10 PCR-DEIA-LiPA25 version 1 method. An event in the multi-site woman-level vaccine efficacy analysis (n = 4,186) was defined as a women with prevalent HPV16/18 DNA at the cervical, anal, or oral regions. Vaccine efficacies (VEs) and 95% confidence intervals (95%CIs) were computed for one-time detection of HPV16/18 in the cervical, anal, and oral regions in this intention-to-treat analysis. Results: Four years following initial vaccination, the combined multi-site woman-level vaccine efficacy against HPV16/18 infections was 64.8%, 95%CI = 54.8-72.8. Multi-site woman-level efficacy was stronger among women without evidence of previous HPV exposure (HPV16/18 seronegative and cervical HPV16/18 DNA negative at enrollment): VE = 83.1%, 95%CI = 72.6-89.6, but was also demonstrated among women with evidence of previous HPV16/18 exposure (HPV16/18 seropositive and cervical HPV16/18 DNA negative at baseline): VE = 49.6%, 95%CI = 2.7-73.9. Further supporting the partial protection of the vaccine in previously HPV16/18-exposed women, we observed a particularly strong vaccine efficacy against HPV16/18 at more than one anatomic site (VE = 91.4%, 95%CI = 81.4-96.6). Indeed, HPV16/18-infected women were significantly less likely to be HPV16/18-infected at two or more anatomic sites in the HPV vaccine arm than the control arm (6 of 81 (7%) vs. 70 of 230 (30%), pCitation Format: Daniel C. Beachler, Aimee R. Kreimer, Mark Schiffman, Rolando Herrero, Sholom Wacholder, Ana Cecilia Rodriguez, Douglas R. Lowy, Carolina Porras, John T. Schiller, Silvia Jimenez, Linda Struijk, John Schussler, Allan Hildesheim, Paula Gonzalez, Costa Rica Vaccine Trial Group. Efficacy of the HPV16/18 vaccine against cervical, anal, and oral HPV infection among women with and without previous HPV16/18 exposure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2015-4680