Intra- and interlaboratory reproducibility evaluation toward international validation status of the AmpFire assay.

To meet the screening goal of WHO's 90-70-90 strategy aimed at eliminating cervical cancer (CC) by 2030, clinical validation of human papillomavirus (HPV) assays is essential to provide accurate and valid results through fulfilling three criteria of the international validation guidelines (IVGs). Previously, the clinical accuracy of the AmpFire® HPV Screening 16/18/HR assay (AmpFire assay) was reported but reproducibility data are lacking. Here, we aim to evaluate the intra- and inter-laboratory reproducibility of the AmpFire assay. The reproducibility of the isothermal AmpFire assay was assessed using 556 cervical cell samples collected from women attending CC screening and biobanked in a Belgian HPV national reference center. This assay detects HPV16, HPV18, and 12 other high-risk HPV (hrHPV) types (31/33/35/39/45/51/52/56/58/59/66/68) in aggregate. Lower 95% confidence interval bound around the assay's reproducibility should exceed 87%, with κ ≥ 0.50. Additionally, a literature review of the assay's clinical performance was performed. The AmpFire assay showed an excellent intralaboratory (96.4%, 95% CI:94.5-97.8%, κ = 0.920) and interlaboratory (95.3%, 95% CI:93.2-96.9%, κ = 0.897) reproducibility. One study demonstrated noninferior sensitivity of a prototype AmpFire assay targeting 15 hrHPV types (including HPV53) to detect CIN2+. However, clinical specificity became similar to the comparator after removing HPV53 from analyses. The low-cost and easy-to-use AmpFire assay presents excellent reproducibility and-after removing HPV53 from the targeted types-fulfills also clinical accuracy requirements. Inclusion of HPV53, which is not recognized as carcinogenic, comprises clinical specificity of screening assays.

Study on the detection rate, genetic polymorphism, viral load, persistent infection capacity, and pathogenicity of human papillomavirus type 81.

Human papillomavirus (HPV) type 81 has recently become one of the most common low-risk HPV types; however, literature focusing on it is limited. This study aimed to analyze the reasons for the increased detection rate of HPV81 and investigate its evolving pathogenicity. We analyzed the detection rates and trends of HPV81 in 229 061 exfoliated cervical cell samples collected from 2014 to 2023; collected samples of HPV81 single infections from two different time periods; and analyzed the allele frequencies, positive selection, viral load, persistent infection capacity, and pathogenicity of E6 and E7 genotypes. We found that the detection rate of HPV81 ranked first among the low-risk types in exfoliated cervical cells and exhibited a significantly increasing trend (p < 0.001). The frequency of the E6 prototype allele of HPV81 (n = 317) was significantly increased (p = 0.018) and demonstrated the strongest adaptive capacity. The viral load and persistent infection capacity of the E6 prototype were significantly higher than those of the mutants, thus serving as key drivers for increasing the detection rate of HPV81 and enhancing its pathogenicity. The viral load was positively correlated with persistent infection capacity and pathogenicity. Persistent infection was a crucial factor in the pathogenicity of HPV81. Successful adaptive evolution of HPV81 is accompanied by enhanced pathogenicity.

Species-level characterization of the cervicovaginal microbiota and its role in human papillomavirus-associated cervical carcinogenesis.

The cervicovaginal microbiome may contribute to human papillomavirus (HPV)-associated cervical carcinogenesis, but studies have been limited by low-resolution analysis methods. Using a high-resolution bioinformatics pipeline, we evaluated the relationship of the cervicovaginal microbiome with HPV and cervical intraepithelial neoplasia (CIN). The cervicovaginal microbiome of 186 women was characterized by sequencing 16S rRNA regions (V3-V4 and V5-V6) and annotated with the high-resolution ANCHOR pipeline. Samples were genotyped for HPV using the Roche-Cobas 4800 assay. We fitted logistic regression models using stepwise forward selection to select species (presence/absence) as correlates of CIN1+ and constructed a linear microbiome-based score using the regression coefficients. An HPV-based score was calculated from a separate logistic regression model to detect CIN1+ . Receiver operating characteristic curve analyses were performed; the area under the curve (AUC) and 95% confidence intervals (CI) were compared between scores. Overall, 66.7% of participants were HPV-positive. 77 unique species were identified: 8 using V3-V4, 48 using V5-V6, and 21 shared. Twelve species were retained via stepwise selection. The AUCs for the microbiome-, and HPV-based scores were 0.7656 (95% CI 0.6885-0.8426), and 0.7529 (95% CI 0.6855-0.8204), respectively. Bacterial species may be involved in cervical carcinogenesis as the microbiome- and HPV-based scores performed similarly for CIN1+ detection.

The prevalence and genotype distribution of human papillomavirus in central Fujian Province during the COVID-19 pandemic.

BACKGROUND: Global human activities were significantly impacted by the emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by the 2019 novel coronavirus. This study aimed to investigate the prevalence and genotype distribution of HPV infection in Central Fujian Province during the pandemic. METHODS: Cervical samples were collected from 21,612 outpatients and 12,664 females who underwent physical examinations and HPV screening at the People's Hospital of Fujian Province in Fuzhou from April 2020 to April 2023. HPV detection and genotyping were conducted using PCR hybridization. RESULTS: The overall HPV infection rate was 16.1% during the COVID-19 pandemic, with the outpatient group exhibiting a greater infection rate (19.0%) than did the healthy group (12.3%). The top five high-risk HPV (HR-HPV) genotypes in both groups were HPV52, HPV53, HPV58, HPV16, and HPV51. Additionally, HPV81 and HPV43 were the two most common low-risk HPV (LR-HPV) genotypes in the patient group, while HPV81 and HPV42 were the two most common LR-HPV genotypes in the healthy group. The highest prevalence of HPV infection was observed in individuals aged ≤ 24 years (28.4%, 95% CI 25.9-30.9), followed by those aged ≥ 55 years (23.6%, 95% CI 21.6-24.7) and other age groups. The prevalence decreased from 23.0% (95% CI 22.4-23.7) in 2018-2019 to 13.8% (95% CI 12.0-15.5) in 2023. CONCLUSION: This study provides valuable insights into the prevalence and genotypes of HPV infection in the female population of Central Fujian Province from 2020 to 2023. The findings indicate that the prevalence of HPV infection in Central Fujian Province remains relatively low compared to the national average. Furthermore, the prevalence of HPV decreased during the COVID-19 pandemic; however, as the pandemic waned, there was potential for an increase in HPV infection rates. Therefore, it is crucial to strengthen HPV screening and vaccination strategies to prevent the potential spread of HPV.

The Prevalence of Epstein-Barr Virus in Normal, Premalignant, and Malignant Uterine Cervical Samples in Iran.

INTRODUCTION: It is suggested that Epstein-Barr virus (EBV) may play an important role in cervical cancer development. Most studies found a higher rate of EBV in cervical cancer samples in comparison to premalignant and normal groups. In this regard, this study aimed to investigate the prevalence of EBV in cervical samples. METHODS: In total, 364 samples from 179 healthy subjects, 124 women with premalignant lesions, and 61 patients with cervical cancer were investigated using nested-PCR. RESULTS: The mean age ± SE was 54.1 ± 13.4 in women with cervical cancer, 36.1 ± 9.4 among women with premalignant lesions, and 36.6 ± 11.5 in healthy individuals. In total, 290 out of 364 samples were human papillomavirus (HPV) positive and the following HPV genotypes were detected among them: HPV 16/18 was found in 43.1%, 23.9%, and 65.5% of normal, premalignant, and malignant samples, respectively, and other high-risk types were detected in 56.9% of normal, 76.1% of premalignant, and 34.5% of malignant samples. The prevalence of EBV was found to be 9.8%, 2.4%, and 2.8% in cervical cancer, premalignant lesions, and normal specimens, respectively, and the difference was statistically significant (p = 0.028). The overall frequency of coinfection between EBV and HPV was shown to be 3.6%. The coinfection was more prevalent among HPV 16/18-infected samples than other high-risk HPVs (6.6 vs. 2.9%) although the difference was not reached a statistically significant difference (p = 0.23). CONCLUSION: Our findings indicated that EBV could play an important role as a cofactor in the progression of cervical cancer. However, future studies with larger sample sizes and the expression analysis of EBV transcripts or proteins are mandatory.

HPV infection and its correlation with p53 and Bcl-2 among pregnant mothers and their infants.

The investigation of perinatal transmission of HPV is vital for early screening of cervical/oral cancers. Here, transmission of HPV from the pregnant women to their infants was studied. p53 and Bcl-2 expressions and their correlations with HPV infection were examined. HPV infection was detected in the cervical and oral swabs of 135 mother-baby pairs employing both PCR and HC-II methods. 1 year follow-up with an interim visit at 3 months for mothers and 6 months for babies was performed. Immunocytochemistry of p53 and Bcl-2 using the streptavidin-biotin peroxidase method was performed. Prevalence of HPV infection in the mothers was 28.14%, (38/135) and 30.37% (41/135) determined by the PCR and HC-II methods respectively. HPV 16 and/or 18 was identified in 81.57% (31/38) and 82.92% (34/41) of the HPV + women estimated by PCR and HC-II methods respectively. Prevalence rate of HPV 16 among the HPV + pregnant women was 63.15% (24/38) and 65.85% (27/41) determined by PCR and HC-II methods respectively. The frequency of perinatal transmission was 21.05% (8/38) and 21.95% (9/41) determined by PCR and HC-II methods respectively at birth. The HPV + infants in the follow up study cleared the infection within 6 weeks. An abnormal nuclear expression of p53 and cytoplasmic expression of Bcl-2 were observed in the HPV + mother-baby pairs. Cesarean section did not protect the infants against perinatal HPV transmission. The detection of p53 and Bcl-2 proteins in the HPV + mother-baby pairs suggests that these biomarkers may be important in the early screening of oral/cervix cancers in positive cases.

Temporal composition of the cervicovaginal microbiome associates with hrHPV infection outcomes in a longitudinal study.

BACKGROUND: Persistent infections with high-risk human papillomavirus (hrHPV) can cause cervical squamous intraepithelial lesions (SIL) that may progress to cancer. The cervicovaginal microbiome (CVM) correlates with SIL, but the temporal composition of the CVM after hrHPV infections has not been fully clarified. METHODS: To determine the association between the CVM composition and infection outcome, we applied high-resolution microbiome profiling using the circular probe-based RNA sequencing technology on a longitudinal cohort of cervical smears obtained from 141 hrHPV DNA-positive women with normal cytology at first visit, of whom 51 were diagnosed by cytology with SIL six months later. RESULTS: Here we show that women with a microbial community characterized by low diversity and high Lactobacillus crispatus abundance at both visits exhibit low risk to SIL development, while women with a microbial community characterized by high diversity and Lactobacillus depletion at first visit have a higher risk of developing SIL. At the level of individual species, we observed that a high abundance for Gardnerella vaginalis and Atopobium vaginae at both visits associate with SIL outcomes. These species together with Dialister micraerophilus showed a moderate discriminatory power for hrHPV infection progression. CONCLUSIONS: Our results suggest that the CVM can potentially be used as a biomarker for cervical disease and SIL development after hrHPV infection diagnosis with implications on cervical cancer prevention strategies and treatment of SIL.

Risk of cervical high-grade squamous intraepithelial neoplasia in cytologic negative and persistently high-risk human papillomavirus positive patients according to genotypes: a retrospective single center analysis.

In January 2020, a different cervical cancer screening program started in Germany. Women above the age of 35 are recommended to have a combined HPV and cytology swab every three years. Showing persistent high-risk human papillomavirus (hrHPV), cytologic negative cervical samples at baseline and after 12 months, patients are referred to colposcopy. Entailing considerable additional workload due to the required colposcopies, we analyzed the risk of high-grade cervical intraepithelial neoplasia (CIN 3) in cytologic negative and persistent hrHPV women according to their hrHPV genotypes.Methods In this single center retrospective study, patients with persistent hrHPV, cytology negative cervical samples from our certified Colposcopy Unit in 2020 and 2021 were analyzed. Patient demographics, hrHPV types, biopsy rates and histological reports were collected.Results During the study, 69 patients were enrolled. Most frequent hrHPV genotypes were: hrHPV other 72.5%; HPV 16, 20.3% and HPV 18, 7.2%. Colposcopy showed no or minor changes in 92.7% and major changes in 7.2%. CIN 3 was found in 7 patients (10.1%). Prevalence of CIN 3 by hrHPV genotypes was 27.3% for HPV16, 20.0% for HPV18 and 7.1% for HPVO. A statistically significant dependency between hrHPV and cervical intraepithelial neoplasia was demonstrated (p = 0.048).Conclusion Within this single center study of persistent hrHPV, cytologic negative samples, patients with HPV 16 were more likely to have high-grade disease compared to other hrHPV subtypes. Larger prospective randomized trials are needed to substantiate our results and obtain adjusted cervical cancer screening time intervals according to the hrHPV genotypes.

Diagnostic cervical excision in patients with HPV positivity, cytological abnormalities- and preoperative cervical stenosis.

AIM: There is currently no protocol for classifying patients with HPV persistence and preoperative stenosis of the cervical canal. This has a significant impact on cytology results, colposcopy results and the possibility of obtaining reliable cervical histology outcomes. Our analysis clearly shows that colposcopy and cytology underestimate the histological results in patients with limited visibility due to the presence of a type 3 transformation zone (TZ). Our analysis revealed a significant discrepancy between the colposcopy and cytology results and the histological outcomes. Insufficient colposcopy led to the underdiagnosis of dysplastic lesions in patients with a type 3 TZ and cervical stenosis. In the case of repeated cytological abnormalities and inadequate colposcopy examination, it is crucial to perform a diagnostic conization to exclude high-grade dysplastic changes and cervical carcinoma. METHODS: We conducted a retrospective analysis of 1,021 conizations performed in tertiary care hospital in Wolfsburg, Germany between 2014 and 2020. Of these surgical procedures, 89 were diagnostic conizations. In our analysis, we defined diagnostic conization as a procedure performed when there is HPV persistence and repeated cytologic abnormalities in combination with a type 3 TZ, and when it is not possible to retrieve a relevant cervical histology sample. RESULTS: In this period, 8.7% of all conizations were diagnostic excisions. We found histological abnormalities in 48 of 89 patients (53.9%). The histological examination of the excised cone revealed high-grade cervical intraepithelial neoplasia (CIN/HSIL) in 9 patients (10.1%) and CIN 2+ (HSIL) in 23 out of the 89 patients (25.8%). Two cases of early-stage cervical carcinoma (FIGO IA1 and FIGO IA2) were confirmed (2.3%). CONCLUSION: Patients with cervical stenosis, high-risk HPV persistence and repeated cytological abnormalities are at high risk of undetected high-grade cervical dysplasia. Histologic confirmation must be ensured in this patient consultation and this can be achieved by performing diagnostic excisions.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

High-Risk Human Papilloma Virus Genotype Distribution and Correlation with Cervical Cytomorphological Data in Turkish and Immigrant Women in Mersin Province.

Human papilloma virus (HPV) is the most common sexually transmitted viral agent in the world and the most common cause of cervical cancer. HPV prevalence and genotype distribution vary by region and demographic data. In a province in the south of Turkey that constantly receives immigration, we aimed to determine the prevalence of high-risk HPV (HR-HPV) genotypes, evaluate the compatibility between cervical Pap smear cytology results patients and HR-HPVs, and make an up-to-date contribution to the elucidation of epidemiological data. In this single-centre study, a total of 12,641 women aged 18 and over were evaluated retrospectively from January 2019 to July 2022. HPV detection and genotyping were analysed by the PCR method. Bethesda scoring was used for Pap smear cytological evaluation. The overall prevalence of HR-HPV was 12.6% (12.7% in Turkish women, 11.2% in foreign women). Among the typed HPVs that were detected, HPV-16 (31%) was found first, followed by HPV-18 (8%). The prevalence of HR-HPV was higher in women with abnormal cytology (977/1762, 55.4%) than in women with normal cytology (620/10879, 5.7%) (p<0.001). Turkey doesn't yet have a national HPV immunisation program. We think that determining the specific regional frequency of other HR-HPVs separately will be useful in the follow-up of the natural course of the type-specific infection and in vaccine studies in the future.

Proof of Concept Study: Comparability of Microbiome Diversity in Self- and Physician-Collected HPV-Positive and HPV-Negative Cervicovaginal Samples.

Recent studies have revealed the impact of human papillomavirus (HPV) infections on the cervicovaginal microbiome; however, few have explored the utility of self-collected specimens (SCS) for microbiome detection, obtained using standardised methods for HPV testing. Here, we present a proof-of-concept analysis utilising Oxford Nanopore sequencing of the 16S rRNA gene in paired samples collected either by the patient using an Evalyn Brush or collected by a physician using liquid-based cytology (LBC). We found no significant differences in the α-diversity estimates between the SCS and LBC samples. Similarly, when analysing ß-diversity, we observed a close grouping of paired samples, indicating that both collection methods detected the same microbiome features. The identification of genera and Lactobacillus species in each sample allowed for their classification into community state types (CSTs). Notably, paired samples had the same CST, while HPV-positive and -negative samples belonged to distinct CSTs. As previously described in other studies, HPV-positive samples exhibited heightened bacterial diversity, reduced Lactobacillus abundance, and an increase in genera like Sneathia or Dialister. Altogether, this study showed comparable results between the SCS and LBC samples, underscoring the potential of self-sampling for analysing the microbiome composition in cervicovaginal samples initially collected for HPV testing in the context of cervical cancer screening.

High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity.

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 µg/mL). We found CSC (10-3 or 10 µg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Vaginal flora in HPV infection: a cross­sectional analysis.

OBJECTIVE: The vaginal flora has been reported to be associated with human papillomavirus (HPV) infection. The purpose of this study was to investigate the characteristics of the cervical microbiota in patients with HPV infection and to analyse the changes in the vaginal flora and enzyme profiles in females with HPV infection. METHODS: We conducted a cross-sectional study involving 206 participants who underwent HPV genotyping, sexually transmitted diseases pathogen testing, cytology examination, and microbiome analysis. Additionally, we collected 115 HPV-negative samples and 48 HPV-positive samples for 16S rRNA amplicon sequencing. The vaginal microbial communities of both groups were analysed for diversity and differences to explore their association with HPV infection. RESULTS: The abundance of Lactobacillus was found to be reduced, while Gardnerella vaginalis was significantly more prevalent in the HPV + group. In terms of alpha diversity indices, the Shannon index (P = .0036) and Simpson index (P = .02) were higher in the HPV + group compared to the HPV - group, indicating greater community diversity in the HPV + group. Among the 10 sexually transmitted diseases pathogens analysed, Uup3 and Uup6 were significantly associated with HPV infection. Statistically significant differences were observed in Nugent scores and bacterial vaginosis between the two groups (P < .05). In functional analysis, 11 proteins and 13 enzymes were found to be significantly altered in the HPV + group. CONCLUSION: Our study demonstrates that disruptions in the vaginal flora are associated with HPV infection. Reduced levels of Lactobacillus, increased prevalence of Gardnerella, and abnormal enzyme profiles are closely linked to HPV infection.

The purpose of this study was to investigate the characteristics of the cervical microbiota in patients with human papillomavirus infection and to analyse the changes in the vaginal flora and enzyme profiles in females with human papillomavirus infection. We conducted a cross-sectional study involving 206 participants who underwent human papillomavirus genotyping, sexually transmitted diseases pathogen testing, cytology examination, and microbiome analysis. Additionally, we collected 115 HPV-negative samples and 48 HPV-positive samples for 16S rRNA amplicon sequencing. The abundance of Lactobacillus was found to be reduced, while Gardnerella vaginalis was significantly more prevalent in the HPV + group. In functional analysis, 11 proteins and 13 enzymes were found to be significantly altered in the HPV + group. Our study demonstrates that disruptions in the vaginal flora are associated with HPV infection. Reduced levels of Lactobacillus, increased prevalence of Gardnerella, and abnormal enzyme profiles are closely linked to HPV infection.

Cervicovaginal Tissue Residence Confers a Distinct Differentiation Program upon Memory CD8 T Cells.

Tissue-resident memory CD8 T cells (CD8 TRM) are critical for maintaining barrier immunity. CD8 TRM have been mainly studied in the skin, lung and gut, with recent studies suggesting that the signals that control tissue residence and phenotype are highly tissue dependent. We examined the T cell compartment in healthy human cervicovaginal tissue (CVT) and found that most CD8 T cells were granzyme B+ and TCF-1- To address if this phenotype is driven by CVT tissue residence, we used a mouse model to control for environmental factors. Using localized and systemic infection models, we found that CD8 TRM in the mouse CVT gradually acquired a granzyme B+, TCF-1- phenotype as seen in human CVT. In contrast to CD8 TRM in the gut, these CD8 TRM were not stably maintained regardless of the initial infection route, which led to reductions in local immunity. Our data show that residence in the CVT is sufficient to progressively shape the size and function of its CD8 TRM compartment.

Cell-penetrating peptide inhibits retromer-mediated human papillomavirus trafficking during virus entry.

Virus replication requires critical interactions between viral proteins and cellular proteins that mediate many aspects of infection, including the transport of viral genomes to the site of replication. In human papillomavirus (HPV) infection, the cellular protein complex known as retromer binds to the L2 capsid protein and sorts incoming virions into the retrograde transport pathway for trafficking to the nucleus. Here, we show that short synthetic peptides containing the HPV16 L2 retromer-binding site and a cell-penetrating sequence enter cells, sequester retromer from the incoming HPV pseudovirus, and inhibit HPV exit from the endosome, resulting in loss of viral components from cells and in a profound, dose-dependent block to infection. The peptide also inhibits cervicovaginal HPV16 pseudovirus infection in a mouse model. These results confirm the retromer-mediated model of retrograde HPV entry and validate intracellular virus trafficking as an antiviral target. More generally, inhibiting virus replication with agents that can enter cells and disrupt essential protein-protein interactions may be applicable in broad outline to many viruses.

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1.

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.

Bacterial vaginosis (BV), a condition in which the vaginal microbiota consists of community of obligate and facultative anaerobes rather than dominated by a single species of Lactobacillus, affects ~30% of women in the US. Women with BV are at 60% increased risk for HIV acquisition and are 3-times more likely to transmit HIV to an uninfected partner. As cervicovaginal mucus (CVM) is the first line of defense against mucosal pathogens and the home of the resident vaginal microbiota, we hypothesized the barrier function of CVM to HIV may be diminished in BV. Here, we characterized CVM properties including pH, lactic acid content, and Nugent score to correlate with the microbiota community composition, which was confirmed by 16S rDNA sequencing on a subset of samples. We then quantified the mobility of fluorescently-labeled HIV virions and nanoparticles to characterize the structural and adhesive barrier properties of CVM. Our analyses included women with Nugent scores categorized as intermediate (4-6) and BV (7-10), women that were either symptomatic or asymptomatic, and a small group of women before and after antibiotic treatment for symptomatic BV. Overall, we found that HIV virions had significantly increased mobility in CVM from women with BV compared to CVM from women with Lactobacillus crispatus-dominant microbiota, regardless of whether symptoms were present. We confirmed using nanoparticles and scanning electron microscopy that the impaired barrier function was due to reduced adhesive barrier properties without an obvious degradation of the physical CVM pore structure. We further confirmed a similar increase in HIV mobility in CVM from women with Lactobacillus iners-dominant microbiota, the species most associated with transitions to BV and that persists after antibiotic treatment for BV. Our findings advance the understanding of the protective role of mucus and highlight the interplay between vaginal microbiota and the innate barrier function mucus.

Cervicovaginal microbiome and natural history of HPV in a longitudinal study.

BACKGROUND: Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections. However, only a small percentage of high-risk (HR) HPV infections progress to cervical precancer and cancer. In this study, we investigated the role of the cervicovaginal microbiome (CVM) in the natural history of HR-HPV. METHODS: This study was nested within the placebo arm of the Costa Rica HPV Vaccine Trial that included women aged 18-25 years of age. Cervical samples from two visits of women with an incident HR-HPV infection (n = 273 women) were used to evaluate the prospective role of the CVM on the natural history of HR-HPV. We focus specifically on infection clearance, persistence, and progression to cervical intraepithelial neoplasia grade 2 and 3 (CIN2+). The CVM was characterized by amplification and sequencing the bacterial 16S V4 rRNA gene region and the fungal ITS1 region using an Illumina MiSeq platform. OTU clustering was performed using QIIME2. Functional groups were imputed using PICRUSt and statistical analyses were performed using R. RESULTS: At Visit 1 (V1) abundance of Lactobacillus iners was associated with clearance of incident HR-HPV infections (Linear Discriminant Analysis (LDA)>4.0), whereas V1 Gardnerella was the dominant biomarker for HR-HPV progression (LDA>4.0). At visit 2 (V2), increased microbial Shannon diversity was significantly associated with progression to CIN2+ (p = 0.027). Multivariate mediation analysis revealed that the positive association of V1 Gardnerella with CIN2+ progression was due to the increased cervicovaginal diversity at V2 (p = 0.040). A full multivariate model of key components of the CVM showed significant protective effects via V1 genus Lactobacillus, OR = 0.41 (0.22-0.79), V1 fungal diversity, OR = 0.90 (0.82-1.00) and V1 functional Cell Motility pathway, OR = 0.75 (0.62-0.92), whereas V2 bacterial diversity, OR = 1.19 (1.03-1.38) was shown to be predictive of progression to CIN2+. CONCLUSION: This study demonstrates that features of the cervicovaginal microbiome are associated with HR-HPV progression in a prospective longitudinal cohort. The analyses indicated that the association of Gardnerella and progression to CIN2+ may actually be mediated by subsequent elevation of microbial diversity. Identified features of the microbiome associated with HR-HPV progression may be targets for therapeutic manipulation to prevent CIN2+. TRIAL REGISTRATION: ClinicalTrials.gov NCT00128661.