A bioinformatic analysis of T-cell epitope diversity in SARS-CoV-2 variants: association with COVID-19 clinical severity in the United States population.

Long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the identification of T-cell epitopes affecting host immunogenicity. In this computational study, we explored the CD8+ epitope diversity estimated in 27 of the most common HLA-A and HLA-B alleles, representing most of the United States population. Analysis of 16 SARS-CoV-2 variants [B.1, Alpha (B.1.1.7), five Delta (AY.100, AY.25, AY.3, AY.3.1, AY.44), and nine Omicron (BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ.1, BQ.1.1, XBB.1, XBB.1.5)] in analyzed MHC class I alleles revealed that SARS-CoV-2 CD8+ epitope conservation was estimated at 87.6%-96.5% in spike (S), 92.5%-99.6% in membrane (M), and 94.6%-99% in nucleocapsid (N). As the virus mutated, an increasing proportion of S epitopes experienced reduced predicted binding affinity: 70% of Omicron BQ.1-XBB.1.5 S epitopes experienced decreased predicted binding, as compared with ~3% and ~15% in the earlier strains Delta AY.100-AY.44 and Omicron BA.1-BA.5, respectively. Additionally, we identified several novel candidate HLA alleles that may be more susceptible to severe disease, notably HLA-A*32:01, HLA-A*26:01, and HLA-B*53:01, and relatively protected from disease, such as HLA-A*31:01, HLA-B*40:01, HLA-B*44:03, and HLA-B*57:01. Our findings support the hypothesis that viral genetic variation affecting CD8 T-cell epitope immunogenicity contributes to determining the clinical severity of acute COVID-19. Achieving long-term COVID-19 immunity will require an understanding of the relationship between T cells, SARS-CoV-2 variants, and host MHC class I genetics. This project is one of the first to explore the SARS-CoV-2 CD8+ epitope diversity that putatively impacts much of the United States population.

The Role of Prevotella Species in Female Genital Tract Infections.

Female genital tract infections (FGTIs) include vaginal infections (e.g., bacterial vaginosis [BV]), endometritis, pelvic inflammatory disease [PID], and chorioamnionitis [amniotic fluid infection]. They commonly occur in women of reproductive age and are strongly associated with multiple adverse health outcomes including increased risk of HIV/sexually transmitted infection acquisition and transmission, infertility, and adverse birth outcomes such as preterm birth. These FGTIs are characterized by a disruption of the cervicovaginal microbiota which largely affects host immunity through the loss of protective, lactic acid-producing Lactobacillus spp. and the overgrowth of facultative and strict anaerobic bacteria. Prevotella species (spp.), anaerobic Gram-negative rods, are implicated in the pathogenesis of multiple bacterial FGTIs. Specifically, P. bivia, P. amnii, and P. timonensis have unique virulence factors in this setting, including resistance to antibiotics commonly used in treatment. Additionally, evidence suggests that the presence of Prevotella spp. in untreated BV cases can lead to infections of the upper female genital tract by ascension into the uterus. This narrative review aims to explore the most common Prevotella spp. in FGTIs, highlight their important role in the pathogenesis of FGTIs, and propose future research in this area.

Microbial interactions among Gardnerella, Prevotella and Fannyhessea prior to incident bacterial vaginosis: protocol for a prospective, observational study.

INTRODUCTION: The aetiology of bacterial vaginosis (BV), a biofilm-associated vaginal infection, remains unknown. Epidemiologic data suggest that it is sexually transmitted. BV is characterised by loss of lactic acid-producing lactobacilli and an increase in facultative and strict anaerobic bacteria. Gardnerella spp are present in 95%-100% of cases; Gardnerella vaginalis has been found to be more virulent than other BV-associated bacteria (BVAB) in vitro. However, G. vaginalis is found in women with normal vaginal microbiota and colonisation is not sufficient for BV development. We hypothesise that Gardnerella spp initiate BV biofilm formation, but incident BV (iBV) requires incorporation of other key BVAB (ie, Prevotella bivia, Fannyhessea vaginae) into the biofilm that alter the transcriptome of the polymicrobial consortium. This study will investigate the sequence of microbiologic events preceding iBV. METHODS AND ANALYSIS: This study will enrol 150 women aged 18-45 years with normal vaginal microbiota and no sexually transmitted infections at a sexual health research clinic in Birmingham, Alabama. Women will self-collect twice daily vaginal specimens up to 60 days. A combination of 16S rRNA gene sequencing, qPCR for Gardnerella spp, P. bivia and F. vaginae, and broad range 16S rRNA gene qPCR will be performed on twice daily vaginal specimens from women with iBV (Nugent score 7-10 on at least 2 consecutive days) and controls (with comparable age, race, contraceptive method and menstrual cycle days) maintaining normal vaginal microbiota to investigate changes in the vaginal microbiota over time for women with iBV. Participants will complete daily diaries on multiple factors including sexual activity. ETHICS AND DISSEMINATION: This protocol is approved by the University of Alabama at Birmingham Institutional Review Board (IRB-300004547) and written informed consent will be obtained from all participants. Findings will be presented at scientific conferences and published in peer-reviewed journals as well as disseminated to providers and patients in communities of interest.

Association of Chlamydia trachomatis burden with the vaginal microbiota, bacterial vaginosis, and metronidazole treatment.

Bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is a common coinfection with Chlamydia trachomatis (Ct), and BV-associated bacteria (BVAB) and their products have been implicated in aiding Ct evade natural immunity. Here, we determined if a non-optimal vaginal microbiota was associated with a higher genital Ct burden and if metronidazole, a standard treatment for BV, would reduce Ct burden or aid in natural clearance of Ct infection. Cervicovaginal samples were collected from women at enrollment and, if testing positive for Ct infection, at a follow-up visit approximately one week later. Cervical Ct burden was assessed by inclusion forming units (IFU) and Ct genome copy number (GCN), and 16S rRNA gene sequencing was used to determine the composition of the vaginal microbiota. We observed a six-log spectrum of IFU and an eight-log spectrum of GCN in our study participants at their enrollment visit, but BV, as indicated by Amsel's criteria, Nugent scoring, or VALENCIA community state typing, did not predict infectious and total Ct burden, although IFU : GCN increased with Amsel and Nugent scores and in BV-like community state types. Ct burden was, however, associated with the abundance of bacterial species in the vaginal microbiota, negatively with Lactobacillus crispatus and positively with Prevotella bivia. Women diagnosed with BV were treated with metronidazole, and Ct burden was significantly reduced in those who resolved BV with treatment. A subset of women naturally cleared Ct infection in the interim, typified by low Ct burden at enrollment and resolution of BV. Abundance of many BVAB decreased, and Lactobacillus increased, in response to metronidazole treatment, but no changes in abundances of specific vaginal bacteria were unique to women who spontaneously cleared Ct infection.

Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring.

Tumor microbiota can produce active metabolites that affect cancer and immune cell signaling, metabolism, and proliferation. Here, we explore tumor and gut microbiome features that affect chemoradiation response in patients with cervical cancer using a combined approach of deep microbiome sequencing, targeted bacterial culture, and in vitro assays. We identify that an obligate L-lactate-producing lactic acid bacterium found in tumors, Lactobacillus iners, is associated with decreased survival in patients, induces chemotherapy and radiation resistance in cervical cancer cells, and leads to metabolic rewiring, or alterations in multiple metabolic pathways, in tumors. Genomically similar L-lactate-producing lactic acid bacteria commensal to other body sites are also significantly associated with survival in colorectal, lung, head and neck, and skin cancers. Our findings demonstrate that lactic acid bacteria in the tumor microenvironment can alter tumor metabolism and lactate signaling pathways, causing therapeutic resistance. Lactic acid bacteria could be promising therapeutic targets across cancer types.

A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities.

Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.

State of the Art for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge among reproductive-age women. It is associated with multiple adverse health outcomes, including increased risk of acquisition of HIV and other sexually transmitted infections (STIs), in addition to adverse birth outcomes. While it is known that BV is a vaginal dysbiosis characterized by a shift in the vaginal microbiota from protective Lactobacillus species to an increase in facultative and strict anaerobic bacteria, its exact etiology remains unknown. The purpose of this minireview is to provide an updated overview of the range of tests currently used for the diagnosis of BV in both clinical and research settings. This article is divided into two primary sections: traditional BV diagnostics and molecular diagnostics. Molecular diagnostic assays, particularly 16S rRNA gene sequencing, shotgun metagenomic sequencing, and fluorescence in situ hybridization (FISH), are specifically highlighted, in addition to multiplex nucleic acid amplification tests (NAATs), given their increasing use in clinical practice (NAATs) and research studies (16S rRNA gene sequencing, shotgun metagenomic sequencing, and FISH) regarding the vaginal microbiota and BV pathogenesis. We also provide a discussion of the strengths and weaknesses of current BV diagnostic tests and discuss future challenges in this field of research.

HPV-related anal cancer is associated with changes in the anorectal microbiome during cancer development.

Background: Squamous cell carcinoma of the anus (SCCA) is a rare gastrointestinal cancer. Factors associated with progression of HPV infection to anal dysplasia and cancer are unclear and screening guidelines and approaches for anal dysplasia are less clear than for cervical dysplasia. One potential contributing factor is the anorectal microbiome. In this study, we aimed to identify differences in anal microbiome composition in the settings of HPV infection, anal dysplasia, and anal cancer in this rare disease. Methods: Patients were enrolled in two prospective studies. Patients with anal dysplasia were part of a cross-sectional cohort that enrolled women with high-grade lower genital tract dysplasia. Anorectal tumor swabs were prospectively collected from patients with biopsy-confirmed locally advanced SCCA prior to receiving standard-of-care chemoradiotherapy (CRT). Patients with high-grade lower genital tract dysplasia without anal dysplasia were considered high-risk (HR Normal). 16S V4 rRNA Microbiome sequencing was performed for anal swabs. Alpha and Beta Diversity and composition were compared for HR Normal, anal dysplasia, and anal cancer. Results: 60 patients with high-grade lower genital tract dysplasia were initially enrolled. Seven patients had concurrent anal dysplasia and 44 patients were considered HR Normal. Anorectal swabs from 21 patients with localized SCCA were included, sequenced, and analyzed in the study. Analysis of weighted and unweighted UniFrac distances demonstrated significant differences in microbial community composition between anal cancer and HR normal (p=0.018). LEfSe identified that all three groups exhibited differential enrichment of specific taxa. Peptoniphilus (p=0.028), Fusobacteria (p=0.0295), Porphyromonas (p=0.034), and Prevotella (p=0.029) were enriched in anal cancer specimens when compared to HR normal. Conclusion: Although alpha diversity was similar between HR Normal, dysplasia and cancer patients, composition differed significantly between the three groups. Increased anorectal Peptoniphilus, Fusobacteria, Porphyromonas, and Prevotella abundance were associated with anal cancer. These organisms have been reported in various gastrointestinal cancers with roles in facilitating the proinflammatory microenvironment and neoplasia progression. Future work should investigate a potential role of microbiome analysis in screening for anal dysplasia and investigation into potential mechanisms of how these microbial imbalances influence the immune system and anal carcinogenesis.

Characterization of Vaginal Microbial Community Dynamics in the Pathogenesis of Incident Bacterial Vaginosis, a Pilot Study.

BACKGROUND: Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). METHODS: A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0-3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7-10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. RESULTS: Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. CONCLUSIONS: This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV.

Impact of testosterone use on the vaginal microbiota of transgender men, including susceptibility to bacterial vaginosis: study protocol for a prospective, observational study.

INTRODUCTION: The effect of testosterone (T) therapy on the vaginal microbiota of transgender men (TGM) is not well characterised, although one cross-sectional study comparing the vaginal microbiota of cisgender women to TGM on T≥1 year found that, in 71% of the TGM, the vaginal microbiota was less likely to be Lactobacillus-dominated and more likely to be enriched with >30 other bacterial species, many associated with bacterial vaginosis (BV). This prospective study aims to investigate changes in the composition of the vaginal microbiota over time in TGM who retain their natal genitalia (ie, vagina) and initiate T. In addition, we will identify changes in the vaginal microbiota preceding incident BV (iBV) in this cohort while investigating behavioural factors, along with hormonal shifts, which may be associated with iBV. METHODS AND ANALYSIS: T-naïve TGM who have not undergone gender-affirming genital surgery with normal baseline vaginal microbiota (ie, no Amsel criteria, normal Nugent Score with no Gardnerella vaginalis morphotypes) will self-collect daily vaginal specimens for 7 days prior to initiating T and for 90 days thereafter. These specimens will be used for vaginal Gram stain, 16S rRNA gene sequencing and shotgun metagenomic sequencing to characterise shifts in the vaginal microbiota over time, including development of iBV. Participants will complete daily diaries on douching, menses and behavioural factors including sexual activity during the study. ETHICS AND DISSEMINATION: This protocol is approved through the single Institutional Review Board mechanism by the University of Alabama at Birmingham. External relying sites are the Louisiana State University Health Sciences Center, New Orleans Human Research Protection Program and the Indiana University Human Research Protection Program. Study findings will be presented at scientific conferences and peer-reviewed journals as well as shared with community advisory boards at participating gender health clinics and community-based organisations servicing transgender people. REGISTRATION DETAILS: Protocol # IRB-300008073.

Microbiome Dynamics During Chemoradiation Therapy for Anal Cancer.

PURPOSE: Patients with localized squamous cell carcinoma of the anus (SCCA) who experience treatment toxicity or recurrences have few therapeutic options. Investigation into the microbiome's influence on treatment toxicity and its potential use as a predictive biomarker could improve these patients' outcomes. Our study presents the first longitudinal characterization of the SCCA tumor microbiome and its associations with treatment-related toxicities. METHODS AND MATERIALS: This prospective cohort study included patients with nonmetastatic SCCA receiving standard-of-care chemoradiation therapy. Anorectal swabs of the tumor site were collected before, during, and after treatment. Patient-reported quality-of-life metrics were collected at similar time points. 16S rRNA gene sequencing was used to perform diversity and taxonomic characterization of the SCCA microbiome. Wilcoxon signed-rank tests were used to compare microbial diversity and abundance over time. Wilcoxon rank-sum tests were used to compare microbial profiles of high and low toxicity groups. RESULTS: Twenty-two patients with SCCA were included in this study with a median age of 58.5 years (range, 39-77 years), and 18 (82%) were women. Alpha diversity remained relatively stable throughout chemoradiation therapy except for decreases in the Observed Features (P = .03) index at week 5 relative to baseline. Tumor microbial compositions changed significantly by the end of treatment (P = .03). Differential enrichment of bacteria at specific time points occurred during treatment, including the enrichment of Clostridia at follow-up (vs week 5, q = 0.019) and Corynebacterium at week 5 (vs baseline, q = 0.015; vs follow-up, q = 0.022). Patients experiencing high toxicity at week 5 had higher relative counts of Clostridia, Actinobacteria, and Clostridiales at baseline (P = .03 for all). CONCLUSIONS: The tumor microbiome changes during and after chemoradiation therapy, and patient-reported toxicity levels are associated with patients' microbial profiles. Further studies into these microbial characterizations and toxicity associations will elucidate the tumor microbiome's role in predicting treatment-related outcomes for patients with SCCA.

Precision Combination Therapies Based on Recurrent Oncogenic Coalterations.

Cancer cells depend on multiple driver alterations whose oncogenic effects can be suppressed by drug combinations. Here, we provide a comprehensive resource of precision combination therapies tailored to oncogenic coalterations that are recurrent across patient cohorts. To generate the resource, we developed Recurrent Features Leveraged for Combination Therapy (REFLECT), which integrates machine learning and cancer informatics algorithms. Using multiomic data, the method maps recurrent coalteration signatures in patient cohorts to combination therapies. We validated the REFLECT pipeline using data from patient-derived xenografts, in vitro drug screens, and a combination therapy clinical trial. These validations demonstrate that REFLECT-selected combination therapies have significantly improved efficacy, synergy, and survival outcomes. In patient cohorts with immunotherapy response markers, DNA repair aberrations, and HER2 activation, we have identified therapeutically actionable and recurrent coalteration signatures. REFLECT provides a resource and framework to design combination therapies tailored to tumor cohorts in data-driven clinical trials and preclinical studies. SIGNIFICANCE: We developed the predictive bioinformatics platform REFLECT and a multiomics- based precision combination therapy resource. The REFLECT-selected therapies lead to significant improvements in efficacy and patient survival in preclinical and clinical settings. Use of REFLECT can optimize therapeutic benefit through selection of drug combinations tailored to molecular signatures of tumors. See related commentary by Pugh and Haibe-Kains, p. 1416. This article is highlighted in the In This Issue feature, p. 1397.

Mycoplasma decontamination in Chlamydia trachomatis culture: a curative approach.

Mycoplasma contamination of cell culture represents a serious problem in research and decontamination from cell-propagated obligate intracellular bacteria has proven challenging. Here, we presented an optimized protocol to remove Mycoplasma from contaminated Chlamydia trachomatis culture. A stepwise procedure of Mycoplasma removal entails (i) incubation in nonionic detergent-containing solution and (ii) separation of viable chlamydial organisms by fluorescence-activated cell sorting (FACS), followed by subcloning using a focus-forming assay. We also adapted a polymerase chain reaction (PCR) assay using paired universal and Mycoplasma-specific primers, which are distinguishable from the C. trachomatis counterparts, in combination with Sanger sequencing to determine the presence of mycoplasmas' 16S rRNA genes. These integrated approaches allow for full removal of Mycoplasma, as verified by the improved PCR assay, without compromising the capacity of viable C. trachomatis to adapt to new infection in epithelial cells. Some pitfalls during the Mycoplasma decontamination process are discussed.

ATR Inhibitor M6620 (VX-970) Enhances the Effect of Radiation in Non-Small Cell Lung Cancer Brain Metastasis Patient-Derived Xenografts.

M6620, a selective ATP-competitive inhibitor of the ATM and RAD3-related (ATR) kinase, is currently under investigation with radiation in patients with non-small cell lung cancer (NSCLC) brain metastases. We evaluated the DNA damage response (DDR) pathway profile of NSCLC and assessed the radiosensitizing effects of M6620 in a preclinical NSCLC brain metastasis model. Mutation analysis and transcriptome profiling of DDR genes and pathways was performed on NSCLC patient samples. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, cell cycle, and DNA damage signaling and repair assays. NSCLC brain metastasis patient-derived xenograft models were used to assess intracranial response and overall survival. In vivo IHC was performed to confirm in vitro results. A significant portion of NSCLC patient tumors demonstrated enrichment of DDR pathways. DDR pathways correlated with lung squamous cell histology; and mutations in ATR, ATM, BRCA1, BRCA2, CHEK1, and CHEK2 correlated with enrichment of DDR pathways in lung adenocarcinomas. M6620 reduced colony formation after radiotherapy and resulted in inhibition of DNA DSB repair, abrogation of the radiation-induced G2 cell checkpoint, and formation of dysfunctional micronuclei, leading to enhanced radiation-induced mitotic death. The combination of M6620 and radiation resulted in improved overall survival in mice compared with radiation alone. In vivo IHC revealed inhibition of pChk1 in the radiation plus M6620 group. M6620 enhances the effect of radiation in our preclinical NSCLC brain metastasis models, supporting the ongoing clinical trial (NCT02589522) evaluating M6620 in combination with whole brain irradiation in patients with NSCLC brain metastases.