Abstract 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.

Author summary Despite being the most common sexually transmitted infection and the causal agent of cervical cancer, it is still not clear why only a small proportion of high-risk HPV (HR-HPV) infections progress to cervical cancer. Our study utilizes longitudinal cervicovaginal samples from a prospective cohort, along with advanced epidemiological modeling and mediation analysis, to investigate the association between the cervicovaginal microbiome (CVM) and progression of an incident HR-HPV infection to cervical precancer. The results of our study suggest a novel association between the effect of Gardnerella and disruption of CVM homeostasis that can influence the pathway of HR-HPV infection progression to cervical precancer. We further show the interplay between several key components of the cervicovaginal microbiome and demonstrate that within the context of HR-HPV natural history the effect of Gardnerella is mediated by increased cervicovaginal bacterial diversity directly preceding the progression of a persistent infection to precancer.

Citation: Usyk M, Zolnik CP, Castle PE, Porras C, Herrero R, Gradissimo A, et al. (2020) Cervicovaginal microbiome and natural history of HPV in a longitudinal study. PLoS Pathog 16(3): e1008376. https://doi.org/10.1371/journal.ppat.1008376 Editor: Guido Silvestri, Emory University, UNITED STATES Received: September 6, 2019; Accepted: February 2, 2020; Published: March 26, 2020 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: Data cannot be shared publicly because of the use of confidential patient data. Data are available from the National Cancer Institute Institutional Data Access / Ethics Committee (contact via BefanoB@imsweb.com) for researchers who meet the criteria for access to confidential data. Funding: The Costa Rica HPV Vaccine Trial is a long-standing collaboration between investigators in Costa Rica and the NCI. The trial is sponsored and funded by the NCI (contract N01-CP-11005), with funding support from the National Institutes of Health Office of Research on Women's Health. GlaxoSmithKline Biologicals (GSK) provided vaccine and support for aspects of the trial associated with regulatory submission needs of the company under a Clinical Trials Agreement (FDA BB-IND 7920) during the four-year, randomized blinded phase of our study. MU, RDB, CPZ and AG were funded by the National Cancer Institute (NCI) (U01 CA78527). The original Costa Rica vaccine trial was sponsored and funded by the NCI (contract N01-CP-11005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal's policy and have the following conflicts: John T. Schiller and Douglas R. Lowy report that they are named inventors on US Government-owned HPV vaccine patents that are licensed to GlaxoSmithKline and Merck and for which the National Cancer Institute receives licensing fees. They are entitled to limited royalties as specified by federal law.

Introduction Persistent cervical infections by high-risk (HR) human papillomavirus (HPV) cause virtually all cervical cancers and their immediate precursor lesions [1]. Most sexually active women have been infected with HPV at some point in their lives and in the vast majority the infection is cleared within a few months [2]. However, a subset of women develop a persistent HPV infection that places them at high risk for cervical precancer and cancer [2–5]. Fig 1 illustrates this paradigm canonically referred to as HPV natural history. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. HPV natural history. The natural history of HR-HPV is depicted. Briefly, an incident HR-HPV infection can occur by entering the basal layer through an epithelial abrasion. Most incidence infections are cleared, however some remain persistent for years and decades. Persistence of a HR-HPV infection combined with known risk factors (e.g., smoking) may allow the persistent HR-HPV infection to progress to precancer (cervical intraepithelial neoplasia, CIN). If the lesion does not regress and the HR-HPV is able to successfully integrate into the host-cell genome, clonal expansion may occur and result in an invasive cancer. https://doi.org/10.1371/journal.ppat.1008376.g001 Non-viral factors (HPV co-factors) associated with the outcomes of HR-HPV infections have not been fully elucidated. While smoking [6–9], hormonal contraceptive use [10, 11], and parity [12] are associated with developing precancer and cancer; systemic and local immune responses are thought to be important for clearance and control of infection (persistence vs. clearance) [13, 14]. In addition, specific host immune regulatory alleles (e.g., human leukocyte antigen) are associated with risk of cervical cancer [15, 16]. The local, cervical microenvironment, including the microbiome, may also influence the natural history of HPV infection [17]. Other studies have recently implicated the microbiome’s role in the natural history of other viral infections [18] and a variety of cancers [19–21]. The cervicovaginal microbiome (CVM) is of particular interested because it has been well characterized and specific features have been associated with gynecologic disease and reproductive health [22–24]. The CVM has been categorized into community state types (CSTs) generally defined by a dominance of a specific Lactobacillus species (Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri or Lactobacillus jensenii), or a state of polymicrobialism [25, 26]. Transitions from Lactobacillus dominated CSTs have been linked to detrimental health outcomes including elevated risks for sexually transmitted infections [27], as well as higher incidences of preterm births [28]. An association between increased CVM diversity and prevalence of HR-HPV infection and/or cervical abnormalities (vs. HPV negative) has been reported in several studies [29–33]. Higher abundance of L. crispatus has been shown to be associated with lower HPV prevalence [34] and increased detection of normal cytology [35]. Long-term use of vaginal probiotics with Lactobacillus spp. has been associated with increase clearance of HPV compared to short-term use [36]. However, evidence is conflicting on the association of CVM diversity and the severity of cervical neoplasia [37–39]. Additionally, most studies looking at the natural history of HPV and the microbiome are cross-sectional and therefore lack the ability to draw potential causal links. For the current study, we leveraged longitudinal data and specimens from the placebo arm of a large randomized HPV vaccine trial [40] to examine the impact of the CVM on the natural history of incident HR-HPV infections to study: 1) progression to cervical precancer, 2) viral persistence, and 3) viral clearance.

Discussion Previous cross-sectional studies analyzing the association between the cervicovaginal microbiome and HPV infection outcomes have consistently identified Gardnerella as a key biomarker associated with CIN2+. This has been reported in studies that utilized both next-generation sequencing [39, 41] and other methods of microbiome analyses [52, 43]. We present data that Gardnerella is in fact associated with CIN2+ lesions, but rather than directly causing the CIN2+ lesion, appears to induce a higher diversity CVM over time as measured at V2, which in turn mediates the observed effect of Gardnerella in HR-HPV disease progression. Although it is not clear how a state of polymicrobialism in the presence of a persistent HR-HPV infection leads to the development of epithelial dysplasia, recent studies on the microbiome’s role in other cancers suggests that the answer lies in the establishment of a microbial microenvironment, perhaps a biofilm. For instance, it has been shown that certain cancers (e.g., colorectal cancer and prostate cancer) have distinct microbial communities at the tumor site that are associated with tumor development [44, 45]. In fact, other data from our lab using cervical biopsy tissue samples indicates that there are distinct microbial differences as cervical cancer progresses to advanced FIGO stages (manuscript in preparation). This idea is further supported by data indicating that cervical precancerous lesions that regress, compared to those that progress to cancer, harbor a different immune microenvironment [46]. The local interplay between the microbiome and the local host immune system may be important to understanding the progression of HR-HPV infection to cervical cancer. The protective microbial biomarkers identified at V1 also suggest an association of the microbiome and host innate and acquired immunity in progression to CIN2+. Specifically, the protective effect of bacterial Cell Motility may be due to the known phenomena of bacterial flagella activating host immunity [47–49]. This local activation may facilitate the innate immune system’s ability to clear an active HPV infection. Such stimulation may be critical in HPV control since cervical lesions have been shown to be associated with local immunosuppression through the reduction of factors such as IL-17 [50]. Despite the presence of studies to support this conjecture it should be noted that this type of immune activation needs to be confirmed with rigorous experimental precision. Gardnerella, as discussed above, continuously emerges as a risk factor for CIN2+ development and progression. Based on our findings and published data, the association may be tied to the ability of Gardnerella to be immunosuppressive in the cervicovaginal region [51]. Whereas, it seems that the presence of commensal bacteria (e.g. Lactobacillus) with the ability to stimulate a local immune response may be contributing factors to the clearance of incident HR-HPV infections. Moreover, the presence of bacteria with immunosuppressive attributes, such as Gardnerella, may promote viral persistence and progression. Alternatively, there may be other explanations for the observed associations between the cervicovaginal microbiome and HPV’s natural history. One possible explanation is a host developed or inherited immune deficiency that is a common cofactor for both cervical cancer progression and microbial diversity. For example, elevation of a particular inflammatory cytokine may be both necessary for successful tumor growth and be a causal factor in increasing vaginal microbial diversity. Such a factor may also explain the consistent identification of Gardnerella, which is commonly identified as a biomarker for increased diversity in the CVM [52] and a risk factor for CIN2+. We have identified distinct microbial biomarkers that either protect, or promote the progression of a HR-HPV infection to CIN2+ lesions. In the context of what is known about the cervicovaginal microbiome, it may be that these factors act to suppress (in the case of progression) or activate (in the case of clearance) a localized immune response, which in turn influences the natural history of a HR-HPV infection. However, additional prospective studies are needed to establish a causal link between the cervicovaginal microbiome, the immune system and the natural history of HPV. Nevertheless, our results suggest a marker for identifying women with persistent HR-HPV infection at risk for progression by monitoring the presence of Gardnerella and subsequent elevation in microbial diversity. If future studies support a causal role of the cervicovaginal microbiome and disease progression, then it might be possible to manipulate the CVM in a manner to activate a local immune response. It is possible that HPV vaccination might influence the CVM and future research will be needed to evaluate such potential changes. The strength of this study includes the prospective design and availability of a longitudinal cohort. In addition we used advanced epidemiological methods in a novel way to investigate potential causative factors in cervical intraepithelial neoplasia. Potential weaknesses in this study include the relatively small sample size, homogeneity of the population and the use of only two time points. In summary, through the use of longitudinal samples from the CVT cohort we investigated and identified key features of the cervicovaginal microbiome potentially associated with progression of HR-HPV infection [28, 53–56] (e.g., Gardnerella and subsequent increase vaginal microbial diversity). Additional studies are required to validate the model proposed in this report.

Materials and methods Clinical trial information The study of cervicovaginal microbiome and HR-HPV natural history was a nested analysis within the previously reported CVT [57] (clinical trials registration NCT00128661). Written informed consent was obtained from all participants in CVT. The trial protocol can be obtained from the original trial publication [57]. Ethics statement All CVT participants were adult women between the ages of 18–25 years. All participants were shown a video describing the study design and were then required to provide written consent to continue participating in the trial. Institutional review board approval was obtained for the informed consent forms at both the NCI and in Costa Rica. Registered with Clinicaltrials.gov NCT00128661 Study population and case definitions Subjects for this nested study were selected from the placebo arm of a community-based clinical trial of the HPV 16/18 vaccine in Costa Rica that had enrolled women 18 to 25 years of age in 2004–2005 [57]. Women with an incident HR-HPV infection (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, or 59) were selected for analysis. Incident infections were classified based on outcomes from the well-established model of HR-HPV natural history including outcomes of clearance, persistence and progression [58]. Specifically, outcomes related to the incident HR-HPV infection included women who developed a CIN2 or CIN3 (CIN2+) lesion (progression), women with an infection for 2 or more years with the same type in the absence of a CIN2+ diagnosis (persistent), or women who cleared their incident HR-HPV infections within 1 year (clearance). This analysis included 273 women of whom all had available samples at V1 (first visit positive for the studied HPV type) and 266 who had a second sample at V2 (for persistent, visit that was positive for the same type and at least 305 days after V1; progression, closest visit before diagnosis of CIN2+; clearance, following visit that was negative for that type); all had clinical follow-up data. Seven women, one with clearance, and six with persistence either did not have an available V2 sample or the sample failed in lab testing. Cervical microbiome characterization DNA samples [59] were shipped to the Burk Lab on dry ice where the microbiome analysis was performed. DNA had been extracted from cervical brush samples by DDL Diagnostic Laboratory (Voorberg, The Netherlands) where they had been tested for HPV as previously described [60]. Laboratory procedures for the microbiome analyses were performed within a hood (AirClean Systems, Creedmoor, NC) in an isolated room to minimize environmental contamination and water-blank negative controls were used throughout the testing. Bacterial DNA was amplified using barcoded-primers 16SV4_515F (GTGYCAGCMGCCGCGGTA) and 16SV4_806R (GGACTACHVGGGTWTCTAAT) that amplify the V4 variable region of the 16S rRNA gene [61]. This region has been demonstrated to accurately amplify and resolve vaginal bacteria [62]. PCR reactions were performed with 17.75 μl of nuclease-free PCR-grade water (Lonza, Rockland, ME), 2.5 μl of 10X Buffer w/ MgCl2 (Affymetrix, Santa Clara, CA), 1 μl of MgCl 2 (25 mM, Affymetrix, Santa Clara, California, USA), 0.5 μl of dNTPs (10 mM, Roche, Basel, Switzerland), 0.25 μl of AmpliTaq Gold DNA Polymerase (5 U/μl, Applied Biosystems, Foster City, California), 0.5 μl of HotStart-IT FideliTaq (2.5 U/μl, Affymetrix, Santa Clara, CA), 1 μl of each primer (5 μM), and 0.5 μl of sample DNA. Thermal cycling conditions consisted of initial denaturation at 95°C for 5 min, followed by 15 cycles at 95°C for 1 min, 55°C for 1 min, and 68°C for 1 min, followed by 15 cycles at 95°C for 1 min, 60°C for 1 min, and 68°C for 1 min, and a final extension for 10 min at 68°C on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The fungal DNA ITS1 region was amplified using barcoded-primers ITS1_48F (ACACACCGCCCGTCGCTACT) and ITS1_217R (TTTCGCTGCGTTCTTCATCG) as previously described [63]. PCR reactions were performed with 8.25 μl of nuclease-free PCR-grade water (Lonza), 2.5 μl of 10X Buffer w/ MgCl 2 (Affymetrix), 1 μl of MgCl 2 (25 mM, Affymetrix), 0.5 μl of dNTPs (10 mM, Roche), 0.25 μl of AmpliTaq Gold DNA Polymerase (5 U/μl, Applied Biosystems), 0.5 μl of HotStart-IT FideliTaq (2.5 U/μl, Affymetrix), 1μl of each primer (5 μM), and 10 μl of sample DNA. Thermal cycling conditions consisted of initial denaturation of 95°C for 3 min, followed by 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 68°C for 2 min, followed by a final extension for 10 min at 68°C on a GeneAmp PCR System 9700 (Applied Biosystems). For both amplicon experiments, 20 negative controls were randomly mixed amongst samples. Negative controls were created using nuclease-free PCR-grade water (Lonza) as described above instead of extracted DNA. Barcoded-PCR products were combined for each amplicon type and the DNA fragments (~356 bp for 16S V4 and 400–600 for ITS1) were isolated by gel purification using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Purified PCR products were quantified using a Qubit 2.0 Fluorometic High Sensitivity dsDNA Assay (Life Technologies, Carlsbad, CA) prior to library construction using a KAPA LTP Library Preparation Kit (Kapa Biosystems, Wilmington, MA). Size integrity of the amplicons was validated with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). High-throughput amplicon sequencing of 2x300 paired-end reads was conducted on an Illumina MiSeq (Illumina, San Diego, CA). Bioinformatics Illumina reads were trimmed to remove bases that had a PHRED score of <25 using prinseq-lite V0.0.4 [64]. Quality trimmed reads were then demultiplexed using Novobarcode [65]. Paired-end reads were joined using PANDAseq with default settings [66]. The merged reads were processed through the VSEARCH quality-filtering pipeline [67] to dereplicate the sequences, reduce noise and remove chimeric reads. For bacterial 16S V4 rRNA gene reads, closed-reference OTU picking was performed using VSEARCH [67] with a custom database that contained sequences from the GreenGenes database [68] the Human Oral Microbiome Database (HOMD) [69] and cervicovaginal microbiome 16S reference sequences retrieved from NCBI [70]. Representative sequences were aligned using PyNAST [71] and taxonomy was assigned using VSEARCH [67]. PICRUSt was used to impute microbial functional gene content and to collapse identified genes into functional pathways [72]. Pathways that were associated with HR-HPV clearance were identified through the use of a generalized linear model (GLM) based on statistical significance (p<0.05) and relative abundance (1% or higher across all reads). For fungal ITS1 reads, open-reference OTU picking was performed using VSEARCH [67] and the UNITE database [73]. Taxonomy of representative fungal sequences was assigned using BLAST [74]. Phyloseq [75] was used to import BIOM data for 16S and ITS assays into R separately, followed by the determination of Shannon and Chao1 alpha diversity. For all analyses, bacterial data was subsampled for 2,500 reads. For fungal analyses subsampling was performed at 500 reads. Biomarker discovery analysis was performed using the LEfSe tool [76]. Linear discriminant analysis (LDA) scores greater than 2.0 are considered to be significant [76]. Microbial community state types (CSTs) were assigned on the basis of hierarchical clustering of the 20 most abundant OTUs. Prior to clustering, OTUs were agglomerated at the species level or the lowest identified taxonomic level. Clustering was performed using the wardD2 algorithm using Euclidian distances. Statistical analysis R v3.4.2 [77] was used for statistical analyses. The Kruskal-Wallis test was used to assess significance of continuous data. Linear regression was used to assess the significance of variables associated with the ordered outcome states of a HR-HPV infection (1). Logistic regression was performed using the GLM function and a binomial family generalized linear model in R. For categorical data, dummy variables were created and each individual factor level was tested in a univariate GLM analysis. Models were adjusted for age, smoking, HPV16 and CSTs. Smoking status was determined through a questionnaire and incorporated into the model as ordered categories: never smoked, former smoker and current smoker [40]. Power of GLM results was computed using the lmSupport package [78]. We performed a statistical mediation analysis to test whether V1 Gardnerella (an independent variable) could be acting by inducing a subsequent elevated microbiome diversity at V2 (mediator variable) that influences the outcome of HR-HPV progression using the package mediation [79]. The outcome model we used was binary clearance/progression. Models were adjusted for age, CST, smoking status and HPV16 infection status. In the results we present the mediation effect (average causal mediation effects (ACME)), which is the total effect of V2 Shannon diversity and V1 Gardnerella minus the direct effect of V1 Gardnerella. Additionally, we estimate the direct of effect (presented using the average direct effect (ADE)) of V1 Gardnerella on the binary outcome clearance/progression, minus the effect of the V2 Shannon diversity mediator; the total effect, which is the sum between the indirect effect of the V2 Shannon diversity and the direct effect of the V1 Gardnerella; and the proportion mediated which is the ratio of the ACME and total effect estimates. Investigators in the International Agency for Research on Cancer/World Health Organization Where authors are identified as personnel of the International Agency for Research on Cancer/ World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization. Investigators in the Costa Rica HPV Vaccine Trial (CVT) group Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José, Costa Rica—Bernal Cortés (specimen and repository manager), Paula González (LTFU: co-principal investigator), Rolando Herrero (CVT: co-principal investigator), Silvia E. Jiménez (trial coordinator), Carolina Porras (co-investigator), Ana Cecilia Rodríguez (co-investigator). United States National Cancer Institute, Bethesda, MD, USA—Allan Hildesheim (co-principal investigator & NCI co-project officer), Aimée R. Kreimer (LTFU: co-principal investigator & NCI co-project officer), Douglas R. Lowy (HPV virologist), Mark Schiffman (CVT: medical monitor & NCI co-project officer), John T. Schiller (HPV virologist), Mark Sherman (CVT: QC pathologist), Sholom Wacholder (statistician). Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA (HPV Immunology Laboratory)—Ligia A. Pinto, Troy J. Kemp Georgetown University, Washington, DC, USA—Mary K. Sidawy (CVT: histopathologist) DDL Diagnostic Laboratory, Netherlands (HPV DNA Testing)—Wim Quint, Leen-Jan van Doorn, Linda Struijk. University of California, San Francisco, CA, USA—Joel M. Palefsky (expert on anal HPV infection and disease diagnosis and management), Teresa M. Darragh (pathologist and clinical management) University of Virginia, Charlottesville, VA, USA—Mark H. Stoler (QC pathologist)

Acknowledgments The NCI and Costa Rica investigators are responsible for the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation of the manuscript. We extend a special thanks to the women of Guanacaste and Puntarenas, Costa Rica, who gave of themselves in participating in this effort. In Costa Rica, we acknowledge the tremendous effort and dedication of the staff involved in this project; we would like to specifically acknowledge the meaningful contributions by Carlos Avila, Loretto Carvajal, Rebeca Ocampo, Cristian Montero, Diego Guillen, Jorge Morales and Mario Alfaro. In the United States, we extend our appreciation to the team from Information Management Services (IMS) responsible for the development and maintenance of the data system used in the trial and who serve as the data management center for this effort, especially Jean Cyr, Julie Buckland, John Schussler, and Brian Befano. We thank Dr. Diane Solomon (CVT: medical monitor & QC pathologist) for her invaluable contributions during the randomized blinded phase of the trial and the design of the LTFU and Nora Macklin (CVT) and Kate Torres (LTFU) for the expertise in coordinating the study. We thank the members of the Data and Safety Monitoring Board charged with protecting the safety and interest of participants during the randomized, blinded phase of our study (Steve Self, Chair, Adriana Benavides, Luis Diego Calzada, Ruth Karron, Ritu Nayar, and Nancy Roach) and members of the external Scientific HPV Working Group who have contributed to the success of our efforts over the years (Joanna Cain and Elizabeth Fontham, Co-Chairs, Diane Davey, Anne Gershon, Elizabeth Holly, Silvia Lara, Henriette Raventós, Wasima Rida, Richard Roden, Maria del Rocío Sáenz Madrigal, Gypsyamber D’Souza, and Margaret Stanley).