Comparing one dose of HPV vaccine in girls aged 9-14 years in Tanzania (DoRIS) with one dose in young women aged 15-20 years in Kenya (KEN SHE): an immunobridging analysis of randomised controlled trials.

BACKGROUND: The first randomised controlled trial of single-dose human papillomavirus (HPV) vaccine efficacy, the Kenya single-dose HPV-vaccine efficacy (KEN SHE) trial, showed greater than 97% efficacy against persistent HPV16 and HPV18 infection at 36 months among women in Kenya. We compared antibody responses after one dose of HPV vaccine in the Dose Reduction Immunobridging and Safety Study (DoRIS), the first randomised trial of the single- dose regimen in girls aged 9-14 years, the target age range for vaccination, with those after one dose of the same vaccine in KEN SHE. METHODS: In the DoRIS trial, 930 girls aged 9-14 years in Tanzania were randomly assigned to one, two, or three doses of the 2-valent vaccine (Cervarix) or the 9-valent vaccine (Gardasil-9). The proportion seroconverting and geometric mean concentrations (GMCs) at month 24 after one dose were compared with those in women aged 15-20 years who were randomly assigned to one dose of the same vaccines at the same timepoint in KEN SHE. Batched samples were tested together by virus-like particle ELISA for HPV16 and HPV18 IgG antibodies. Non-inferiority of GMC ratios (DoRIS trial:KEN SHE) was predefined as a lower bound of the 95% CI less than 0·50. FINDINGS: Month 24 HPV16 and HPV18 antibody GMCs in DoRIS were similar or higher than those in KEN SHE. 2-valent GMC ratios were 0·90 (95% CI 0·72-1·14) for HPV16 and 1·02 (0·78-1·33) for HPV18. 9-valent GMC ratios were 1·44 (95% CI 1·14-1·82) and 1·47 (1·13-1·90), respectively. Non-inferiority of antibody GMCs and seropositivity was met for HPV16 and HPV18 for both vaccines. INTERPRETATION: HPV16 and HPV18 immune responses in young girls 24 months after a single dose of 2-valent or 9-valent HPV vaccine were comparable to those in young women who were randomly assigned to a single dose of the same vaccines and in whom efficacy had been shown. A single dose of HPV vaccine, when given to girls in the target age range for vaccination, induces immune responses that could be effective against persistent HPV16 and HPV18 infection at least two years after vaccination. FUNDING: The UK Department of Health and Social Care, the Foreign, Commonwealth, & Development Office, the Global Challenges Research Fund, the UK Medical Research Council and Wellcome Trust Joint Global Health Trials scheme, the Bill and Melinda Gates Foundation, the US National Cancer Institute; the US National Institutes of Health, and the Francis and Dorothea Reed Endowed Chair in Infectious Diseases. TRANSLATION: For the KiSwahili translation of the abstract see Supplementary Materials section.

Costs of delivering human papillomavirus vaccination using a one- or two-dose strategy in Tanzania.

OBJECTIVE: As part of the Dose Reduction Immunobridging and Safety Study of Two HPV Vaccines in Tanzanian Girls (DoRIS; NCT02834637), the current study is one of the first to evaluate the financial and economic costs of the national rollout of an HPV vaccination program in school-aged girls in sub-Saharan Africa and the potential costs associated with a single dose HPV vaccine program, given recent evidence suggesting that a single dose may be as efficacious as a two-dose regimen. METHODS: The World Health Organization's (WHO) Cervical Cancer Prevention and Control Costing (C4P) micro-costing tool was used to estimate the total financial and economic costs of the national vaccination program from the perspective of the Tanzanian government. Cost data were collected in 2019 via surveys, workshops, and interviews with local stakeholders for vaccines and injection supplies, microplanning, training, sensitization, service delivery, supervision, and cold chain. The cost per two-dose and one-dose fully immunized girl (FIG) was calculated. RESULTS: The total financial and economic costs were US$10,117,455 and US$45,683,204, respectively, at a financial cost of $5.17 per two-dose FIG, and an economic cost of $23.34 per FIG. Vaccine and vaccine-related costs comprised the largest proportion of costs, followed by service delivery. In a one-dose scenario, the cost per FIG reduced to $2.51 (financial) and $12.18 (economic), with the largest reductions in vaccine and injection supply costs, and service delivery. CONCLUSIONS: The overall cost of Tanzania's HPV vaccination program was lower per vaccinee than costs estimated from previous demonstration projects in the region, especially in a single-dose scenario. Given the WHO Strategic Advisory Group of Experts on Immunization's recent recommendation to update dosing schedules to either one or two doses of the HPV vaccine, these data provide important baseline data for Tanzania and may serve as a guide for improving coverage going forward. The findings may also aid in the prioritization of funding for countries that have not yet added HPV vaccines to their routine immunizations.

Immunogenicity and safety of one-dose human papillomavirus vaccine compared with two or three doses in Tanzanian girls (DoRIS): an open-label, randomised, non-inferiority trial.

BACKGROUND: An estimated 15% of girls aged 9-14 years worldwide have been vaccinated against human papillomavirus (HPV) with the recommended two-dose or three-dose schedules. A one-dose HPV vaccine schedule would be simpler and cheaper to deliver. We report immunogenicity and safety results of different doses of two different HPV vaccines in Tanzanian girls. METHODS: In this open-label, randomised, phase 3, non-inferiority trial, we enrolled healthy schoolgirls aged 9-14 years from Government schools in Mwanza, Tanzania. Eligible participants were randomly assigned to receive one, two, or three doses of either the 2-valent vaccine (Cervarix, GSK Biologicals, Rixensart) or the 9-valent vaccine (Gardasil-9, Sanofi Pasteur MSD, Lyon). The primary outcome was HPV 16 specific or HPV 18 specific seropositivity following one dose compared with two or three doses of the same HPV vaccine 24 months after vaccination. Safety was assessed as solicited adverse events up to 30 days after each dose and unsolicited adverse events up to 24 months after vaccination or to last study visit. The primary outcome was done in the per-protocol population, and safety was analysed in the total vaccinated population. This study was registered in ClinicalTrials.gov, NCT02834637. FINDINGS: Between Feb 23, 2017, and Jan 6, 2018, we screened 1002 girls for eligibility. 72 girls were excluded. 930 girls were enrolled and randomly assigned to receive one dose of Cervarix (155 participants), two doses of Cervarix (155 participants), three doses of Cervarix (155 participants), one dose of Gardasil-9 (155 participants), two doses of Gardasil-9 (155 participants), or three doses of Gardasil-9 (155 participants). 922 participants received all scheduled doses within the defined window (three withdrew, one was lost to follow-up, and one died before completion; two received their 6-month doses early, and one received the wrong valent vaccine in error; all 930 participants were included in the total vaccinated cohort). Retention at 24 months was 918 (99%) of 930 participants. In the according-to-protocol cohort, at 24 months, 99% of participants who received one dose of either HPV vaccine were seropositive for HPV 16 IgG antibodies, compared with 100% of participants who received two doses, and 100% of participants who received three doses. This met the prespecified non-inferiority criteria. Anti-HPV 18 seropositivity at 24 months did not meet non-inferiority criteria for one dose compared to two doses or three doses for either vaccine, although more than 98% of girls in all groups had HPV 18 antibodies. 53 serious adverse events (SAEs) were experienced by 42 (4·5%) of 930 girls, the most common of which was hospital admission for malaria. One girl died of malaria. Number of events was similar between groups and no SAEs were considered related to vaccination. INTERPRETATION: A single dose of the 2-valent or 9-valent HPV vaccine in girls aged 9-14 years induced robust immune responses up to 24 months, suggesting that this reduced dose regimen could be suitable for prevention of HPV infection among girls in the target age group for vaccination. FUNDING: UK Department for International Development/UK Medical Research Council/Wellcome Trust Joint Global Health Trials Scheme, The Bill & Melinda Gates Foundation, and the US National Cancer Institute. TRANSLATION: For the KiSwahili translation of the abstract see Supplementary Materials section.

Comparing one dose of HPV vaccine in girls aged 9-14 years in Tanzania (DoRIS) with one dose of HPV vaccine in historical cohorts: an immunobridging analysis of a randomised controlled trial.

BACKGROUND: Human papillomavirus (HPV) vaccines are given as a two-dose schedule in children aged 9-14 years, or as three doses in older individuals. We compared antibody responses after one dose of HPV vaccine in the Dose Reduction Immunobridging and Safety Study (DoRIS), a randomised trial of different HPV vaccine schedules in Tanzania, to those from two observational HPV vaccine trials that found high efficacy of one dose up to 11 years against HPV16 and HPV18 (Costa Rica Vaccine Trial [CVT] and Institutional Agency for Research on Cancer [IARC] India trial). METHODS: In this immunobridging analysis of an open-label randomised controlled trial, girls were recruited from 54 government schools in Mwanza, Tanzania, into the DoRIS trial. Girls were eligible if they were aged 9-14 years, healthy, and HIV negative. Participants were randomly assigned (1:1:1:1:1:1), using permutated block sizes of 12, 18, and 24, to one, two, or three doses of the 2-valent vaccine (Cervarix, GSK Biologicals, Rixensart, Belgium) or the 9-valent vaccine (Gardasil 9, Sanofi Pasteur MSD, Lyon, France). For this immunobridging analysis, the primary objective was to compare geometric mean concentrations (GMCs) at 24 months after one dose in the per-protocol population compared with in historical cohorts: the one-dose 2-valent vaccine group in DoRIS was compared with recipients of the 2-valent vaccine Cervarix from CVT and the one-dose 9-valent vaccine group in DoRIS was compared with recipients of the 4-valent vaccine Gardasil (Merck Sharp & Dohme, Whitehouse Station, NJ, USA) from the IARC India trial. Samples were tested together with virus-like particle ELISA for HPV16 and HPV18 IgG antibodies. Non-inferiority of GMC ratios (DoRIS trial vs historical cohort) was predefined as when the lower bound of the 95% CI was greater than 0·50. This study is registered with ClinicalTrials.gov, NCT02834637. FINDINGS: Between Feb 23, 2017, and Jan 6, 2018, we screened 1002 girls for eligibility, of whom 930 were enrolled into DoRIS and 155 each were assigned to one dose, two doses, or three doses of 2-valent vaccine, or one dose, two doses, or three doses of 9-valent vaccine. 154 (99%) participants in the one-dose 2-valent vaccine group (median age 10 years [IQR 9-12]) and 152 (98%) in the one-dose 9-valent vaccine group (median age 10 years [IQR 9-12]) were vaccinated and attended the 24 month visit, and so were included in the analysis. 115 one-dose recipients from the CVT (median age 21 years [19-23]) and 139 one-dose recipients from the IARC India trial (median age 14 years [13-16]) were included in the analysis. At 24 months after vaccination, GMCs for HPV16 IgG antibodies were 22·9 international units (IU) per mL (95% CI 19·9-26·4; n=148) for the DoRIS 2-valent vaccine group versus 17·7 IU/mL (13·9-22·5; n=97) for the CVT (GMC ratio 1·30 [95% CI 1·00-1·68]) and 13·7 IU/mL (11·9-15·8; n=145) for the DoRIS 9-valent vaccine group versus 6·7 IU/mL (5·5-8·2; n=131) for the IARC India trial (GMC ratio 2·05 [1·61-2·61]). GMCs for HPV18 IgG antibodies were 9·9 IU/mL (95% CI 8·5-11·5: n=141) for the DoRIS 2-valent vaccine group versus 8·0 IU/mL (6·4-10·0; n=97) for the CVT trial (GMC ratio 1·23 [95% CI 0·95-1·60]) and 5·7 IU/mL (4·9-6·8; n=136) for the DoRIS 9-valent vaccine group versus 2·2 IU/mL (1·9-2·7; n=129) for the IARC India trial (GMC ratio 2·12 [1·59-2·83]). Non-inferiority of antibody GMCs was met for each vaccine for both HPV16 and HPV18. INTERPRETATION: One dose of HPV vaccine in young girls might provide sufficient protection against persistent HPV infection. A one-dose schedule would reduce costs, simplify vaccine delivery, and expand access to the vaccine. FUNDING: UK Department for International Development/UK Medical Research Council/Wellcome Trust Joint Global Health Trials Scheme, The Bill & Melinda Gates Foundation, and the US National Cancer Institute. TRANSLATION: For the KiSwahili translation of the abstract see Supplementary Materials section.

A novel MBTPS2 variant associated with BRESHECK syndrome impairs sterol-regulated transcription and the endoplasmic reticulum stress response.

Ichthyosis follicularis, atrichia, and photophobia syndrome (IFAP syndrome) is a rare, X-linked disorder caused by pathogenic variants in membrane-bound transcription factor protease, site 2 (MBTPS2). Pathogenic MBTPS2 variants also cause BRESHECK syndrome, characterized by the IFAP triad plus intellectual disability and multiple congenital anomalies. Here we present a patient with ichthyosis, sparse hair, pulmonic stenosis, kidney dysplasia, hypospadias, growth failure, thrombocytopenia, anemia, bone marrow fibrosis, and chronic diarrhea found by research-based exome sequencing to harbor a novel, maternally inherited MBTPS2 missense variant (c.766 G>A; (p.Val256Leu)). In vitro modeling supports variant pathogenicity, with impaired cell growth in cholesterol-depleted media, attenuated activation of the sterol regulatory element-binding protein pathway, and failure to activate the endoplasmic reticulum stress response pathway. Our case expands both the genetic and phenotypic spectrum of BRESHECK syndrome to include a novel MBTPS2 variant and cytopenias, bone marrow fibrosis, and chronic diarrhea.

Adolescent Health Series: HPV infection and vaccination in sub-Saharan Africa: 10 years of research in Tanzanian female adolescents - narrative review.

Cervical cancer is the leading cause of cancer-related morbidity and mortality in many sub-Saharan African (SSA) countries, including Tanzania. Most cervical cancer cases worldwide are attributable to infection of the cervix with Human Papillomavirus (HPV), a vaccine-preventable sexually transmitted infection (STI). Over the past 10 years, we have conducted a programme of HPV research in pre-adolescents and adolescents in Mwanza, the second-largest city in Tanzania, which is situated in a malaria-endemic region. In this narrative review article, we summarise the contribution of our work, alongside work of others, to improve the understanding of HPV epidemiology in SSA and development of setting-appropriate, evidence-based intervention strategies. We present evidence for very high prevalence and incidence of HPV infection among female SSA adolescents around the time of sexual debut, describe risk factors for HPV acquisition, and discuss associations between HPV, HIV and other STIs, which are also highly prevalent within this population. We summarise findings from early clinical trials of HPV vaccines in SSA, the first of which was an immunogenicity and safety trial conducted in Mwanza, Tanzania, and Dakar, Senegal. Within the trial, we evaluated for the first time the potential impact of malaria and helminth infection on vaccine-induced antibody responses in Tanzanian girls. We describe research evaluating optimal HPV vaccine delivery strategies within this setting, perceived requirements for and barriers to vaccine implementation among key informants from LMIC, vaccine acceptability among girls and parents, and opportunities for co-delivery of interventions alongside HPV vaccination to an adolescent population. Finally, we discuss country-level barriers to vaccine uptake in LMIC, and ongoing studies in Tanzania and other SSA countries of reduced-dose HPV vaccination schedules that may alleviate cost and logistical barriers to vaccine implementation.

Rate of thrombosis in children and adolescents hospitalized with COVID-19 or MIS-C.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with thrombotic complications in adults, but the incidence of COVID-19-related thrombosis in children and adolescents is unclear. Most children with acute COVID-19 have mild disease, but coagulopathy has been associated with multisystem inflammatory syndrome in children (MIS-C), a postinfectious complication. We conducted a multicenter retrospective cohort study to determine the incidence of thrombosis in children hospitalized with COVID-19 or MIS-C and evaluate associated risk factors. We classified patients into 1 of 3 groups for analysis: COVID-19, MIS-C, or asymptomatic SARS-CoV-2. Among a total of 853 admissions (COVID-19, n = 426; MIS-C, n = 138; and asymptomatic SARS-CoV-2, n = 289) in 814 patients, there were 20 patients with thrombotic events (TEs; including 1 stroke). Patients with MIS-C had the highest incidence (9 [6.5%] of 138) vs COVID-19 (9 [2.1%] of 426) or asymptomatic SARS-CoV-2 (2 [0.7%] of 289). In patients with COVID-19 or MIS-C, a majority of TEs (89%) occurred in patients age ≥12 years. Patients age ≥12 years with MIS-C had the highest rate of thrombosis at 19% (9 of 48). Notably, 71% of TEs that were not present on admission occurred despite thromboprophylaxis. Multivariable analysis identified the following as significantly associated with thrombosis: age ≥12 years, cancer, presence of a central venous catheter, and MIS-C. In patients with COVID-19 or MIS-C, hospital mortality was 2.3% (13 of 564), but it was 28% (5 of 18) in patients with TEs. Our findings may help inform pediatric thromboprophylaxis strategies.

A dose-reduction HPV vaccine immunobridging trial of two HPV vaccines among adolescent girls in Tanzania (the DoRIS trial) - Study protocol for a randomised controlled trial.

BACKGROUND: Human papillomavirus (HPV) infection is the primary cause of cervical cancer. In 2018, the World Health Organization (WHO) Director General announced his commitment to eliminate cervical cancer, with HPV vaccination as a priority. However, the costs of setting up a multi-dose HPV vaccination programme remain a barrier to its introduction. METHODS/DESIGN: We are conducting a randomised-controlled trial of reduced dose schedules of HPV vaccine in Tanzania to establish whether a single dose produces immune responses that will be effective in preventing cervical cancer. 930 girls aged 9-14 years in Mwanza, Tanzania, were randomised to one of 6 arms, comprising 3 different dose schedules of the 2-valent (Cervarix) and 9-valent (Gardasil-9) HPV vaccines: 3 doses; 2 doses given 6 months apart; or a single dose. All participants will be followed for 36 months; those in the 1 and 2 dose arms will be followed for 60 months. Trial outcomes focus on vaccine immune responses including HPV 16/18-specific antibody levels, antibody avidity, and memory B cell responses. Results will be immunobridged to historical cohorts of girls and young women in whom efficacy has been demonstrated. DISCUSSION: This is the first randomised trial of the single dose HPV vaccine schedule in the target age group. The trial will allow us to examine the quality and durability of immune responses of reduced dose schedules in a population with high burden of malaria and other infections that may affect vaccine immune responses. Initial results (24 months) are expected to be published in early 2021.

Efficacy and immunogenicity of a single dose of human papillomavirus vaccine compared to no vaccination or standard three and two-dose vaccination regimens: A systematic review of evidence from clinical trials.

OBJECTIVES: This study aimed to systematically review the literature on the efficacy and immunogenicity of single-dose HPV vaccination compared to no vaccination or multi-dose schedules among vaccine trial participants. METHODS: Medline, EMBASE, Global Health Database and Cochrane Central Register of Controlled Trials were searched for publications and conference abstracts (dated January 1999-August 2018) using MeSH and non-MeSH terms for human papillomavirus AND vaccines AND (immunogenicity OR efficacy/effectiveness) AND dosage. Search results were screened against pre-specified eligibility criteria. Data were extracted from included articles, and a narrative synthesis conducted on efficacy against HPV16/18 infection and humoral immunogenicity. RESULTS: Seven of 6,523 unique records identified were included in the review. Six were nested observational studies of participants randomised to receive two or three doses in three large HPV vaccine trials, in which some participants did not complete their allocated schedules. One small pilot study prospectively allocated participants to receive one or no vaccine dose. Frequency of HPV16/18 infection was low (e.g. <1% for 12-month-persistent infection) in all vaccinated participants up to seven years post vaccination and did not significantly differ by number of doses (p > 0.05 in all cases). Frequency of infection was significantly lower in one-dose recipients compared to unvaccinated controls (p < 0.01 for all infection endpoints in each study). HPV16/18 seropositivity rates were high in all HPV vaccine recipients (100% in three of four studies reporting this endpoint), though antibody levels were lower with one compared to two or three doses. CONCLUSIONS: This review supports the premise that one HPV vaccine dose may be as effective in preventing HPV infection as multi-dose schedules in healthy young women. However, it also highlights the paucity of available evidence from purpose-designed, prospectively-randomised trials. Results from ongoing clinical trials assessing the efficacy and immunogenicity of single-dose HPV vaccination compared to currently-recommended schedules are awaited.

Discovery of a novel long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth.

BACKGROUND: Virtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA. METHODS: Rapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3'UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth. RESULTS: In this study, a previously unannotated lncRNA lying within exon six and 3'UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA "HULLK" for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation. CONCLUSIONS: HULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa.

Stratification of Latent Mycobacterium tuberculosis Infection by Cellular Immune Profiling.

Background: Recently acquired and remotely acquired latent Mycobacterium tuberculosis infection (LTBI) are clinically indistinguishable, yet recent acquisition of infection is the greatest risk factor for progression to tuberculosis in immunocompetent individuals. We aimed to evaluate the ability of cellular immune signatures that differ between active tuberculosis and LTBI to distinguish recently from remotely acquired LTBI. Methods: Fifty-nine individuals were recruited: 20 had active tuberculosis, 19 had recently acquired LTBI, and 20 had remotely acquired LTBI. The proportion of mycobacteria-specific CD4+ T cells secreting tumor necrosis factor α (TNF-α) but not interferon γ or interleukin 2 which had a differentiated effector phenotype (TNF-α-only TEFF), and the level of CD27 expression on IFN-γ-producing CD4+ T cells, were detected by flow cytometry. Results: The TNF-α-only TEFF signature was significantly higher in the group with recently acquired LTBI, compared with the group with remotely acquired LTBI (P < .0001), and it discriminated between these groups with high sensitivity and specificity, with an area under the curve of 0.87. Two signatures incorporating CD27 expression did not distinguish between recently and remotely acquired LTBI. Interestingly, the TNF-α-only TEFF signature in participants with recently acquired LTBI was more similar to that in participants with tuberculosis than that in participants with remotely acquired LTBI, suggesting that recently acquired LTBI is immunologically more similar to tuberculosis than remotely acquired LTBI. Conclusions: These findings reveal marked biological heterogeneity underlying the clinically homogeneous phenotype of LTBI, providing a rationale for immunological risk stratification to improve targeting of LTBI treatment.

T-cell immunophenotyping distinguishes active from latent tuberculosis.

BACKGROUND: Changes in the phenotype and function of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4+ and CD8+ T-cell subsets in response to stage of infection may allow discrimination between active tuberculosis and latent tuberculosis infection. METHODS: A prospective comparison of M. tuberculosis-specific cellular immunity in subjects with active tuberculosis and latent tuberculosis infection, with and without human immunodeficiency virus (HIV) coinfection. Polychromatic flow cytometry was used to measure CD4+ and CD8+ T-cell subset phenotype and secretion of interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α). RESULTS: Frequencies of CD4+ and CD8+ cells secreting IFN-γ-only, TNF-α-only and dual IFN-γ/TNF-α were greater in active tuberculosis vs latent tuberculosis infection. All M. tuberculosis-specific CD4+ subsets, with the exception of IL-2-only cells, switched from central to effector memory phenotype in active tuberculosis vs latent tuberculosis infection, accompanied by a reduction in IL-7 receptor α (CD127) expression. The frequency of PPDspecific CD4+ TNF-α-only-secreting T cells with an effector phenotype accurately distinguished active tuberculosis from latent tuberculosis infection with an area under the curve of 0.99, substantially more discriminatory than measurement of function alone. CONCLUSIONS: Combined measurement of T-cell phenotype and function defines a highly discriminatory biomarker of tuberculosis disease activity. Unlocking the diagnostic and monitoring potential of this combined approach now requires validation in large-scale prospective studies.

Identification of kinases regulating prostate cancer cell growth using an RNAi phenotypic screen.

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer.