Clinical SARS-CoV-2 Kinetic Profiles Are Dependent on the Viral Strain and Host Vaccination Status.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection kinetics in a real-world, clinical setting represent a knowledge gap in understanding the underlying coronavirus disease 2019 (COVID-19) pathogenesis. There are scant reports of the dynamics describing the two principal components of the viral life cycle, namely, the rapid proliferation and slower clearance phases. Here, we present results from an ongoing workplace clinical surveillance study during which two vaccinated participants became infected with SARS-CoV-2 Omicron variant (BA.1. lineage). The subjects were followed longitudinally with high temporal resolution, allowing the kinetics of both viral phases to be characterized. The viral doubling times in the proliferation phase (3.3 to 3.5 h) and maximum measured viral loads were similar to those observed for unvaccinated individuals infected with an earlier SARS-CoV-2 strain. However, the clearance phase was much shorter in the current study and unexpectedly displayed a multimodal profile. Longitudinal whole-genome SARS-CoV-2 sequencing identified a stable mutation that arose in one of the participants over the 2-week period of positivity. Our small study provides rare insight into the clinical SARS-CoV-2 dynamics, with significance for public health measures and the biology underlying COVID-19. IMPORTANCE We are conducting an ongoing SARS-CoV-2 workplace clinical study based on frequent, longitudinal disease surveillance of staff and household members. Here, we investigated the viral dynamics in two recently vaccinated participants who became infected with the same Omicron variant of SARS-CoV-2. Because the subjects were enrolled in our study, we were able to track the entire viral life cycle with high temporal resolution, with samples collected every 12 h. Surprisingly, the short viral proliferation phase and maximum viral loads in nasal swab samples were similar to our previous observations with unvaccinated participants and an earlier viral strain. However, the decay phase, indicative of viral clearance, was much shorter here. Our results provide a rare, real-world glimpse of the clinical SARS-CoV-2 replication kinetics, potentially impacting immediate therapies and awareness of earlier and greater transmission potential.

Early SARS-CoV-2 dynamics and immune responses in unvaccinated participants of an intensely sampled longitudinal surveillance study.

Background: A comprehensive understanding of the SARS-CoV-2 infection dynamics and the ensuing host immune responses is needed to explain the pathogenesis as it relates to viral transmission. Knowledge gaps exist surrounding SARS-CoV-2 in vivo kinetics, particularly in the earliest stages after exposure. Methods: An ongoing, workplace clinical surveillance study was used to intensely sample a small cohort longitudinally. Nine study participants who developed COVID-19 between November, 2020 and March, 2021 were monitored at high temporal resolution for three months in terms of viral loads as well as associated inflammatory biomarker and antibody responses. CD8 + T cells targeting SARS-CoV-2 in blood samples from study participants were evaluated. Results: Here we show that the resulting datasets, supported by Bayesian modeling, allowed the underlying kinetic processes to be described, yielding a number of unexpected findings. Early viral replication is rapid (median doubling time, 3.1 h), providing a narrow window between exposure and viral shedding, while the clearance phase is slow and heterogeneous. Host immune responses different widely across participants. Conclusions: Results from our small study give a rare insight into the life-cycle of COVID-19 infection and hold a number of important biological, clinical, and public health implications.

A Randomized, Open-Label, Crossover Phase 1 Safety and Pharmacokinetic Study of Oral Maraviroc and Maraviroc 1% Gel (the CHARM-03 Study).

The Combination HIV Antiretroviral Rectal Microbicide-3 (CHARM-03) study was a randomized, open-label, crossover Phase 1 safety and pharmacokinetic (PK) study of oral maraviroc (MVC) and MVC 1% gel. At a single site, healthy HIV-uninfected men and women were enrolled and randomized to an open label crossover sequence of eight consecutive daily exposures to MVC 300 mg dosed orally, MCV 1% gel dosed rectally, and MVC 1% gel dosed vaginally. Male participants received oral and rectal dosing and female participants received oral, rectal, and vaginal dosing. Assessments were undertaken at baseline and following each 8-day period and included collection of plasma, rectal/cervical tissue (CT), and rectal/endocervical/vaginal fluids. Eleven men and nine women were enrolled. Two participants withdrew from the study before receiving study product. There were 25 adverse events, of which 24 were Grade 1 (G1) and one was G2 (unrelated). After eight doses, MVC was quantifiable in all samples following oral, rectal, or vaginal product administration. The highest drug concentrations in plasma, rectal tissue (RT), and CT were associated with oral, rectal, and vaginal drug delivery, respectively. There were significant reductions in tissue drug concentrations when rectal and cervical biopsies were incubated in media before tissue processing for PK (p < .0001). Only oral MVC was associated with limited protection in the rectal explant HIV challenge model (p < .05). There were no immunological changes in RT, and all products were acceptable to participants. In conclusion, all products were found to be safe and acceptable and did not induce local inflammation. The lack of ex vivo efficacy demonstrated in study samples may be due to rapid disassociation of MVC from the explant tissue. ClinicalTrials.gov Identifier: NCT02346084.

Longitudinal COVID-19 Surveillance and Characterization in the Workplace with Public Health and Diagnostic Endpoints.

Public health practices and high vaccination rates currently represent the primary interventions for managing the spread of coronavirus disease 2019 (COVID-19). We initiated a clinical study based on frequent, longitudinal workplace disease surveillance to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission among employees and their household members. We hypothesized that the study would reduce the economic burden and loss of productivity of both individuals and small businesses resulting from standard isolation methods, while providing new insights into virus-host dynamics. Study participants (27 employees and 27 household members) consented to provide frequent nasal or oral swab samples that were analyzed by reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 RNA. Two study participants were found to be infected by SARS-CoV-2 during the study. One subject, a household member, was SARS-CoV-2 RNA positive for at least 71 days and had quantifiable serum virus-specific antibody concentrations for over 1 year. One unrelated employee became positive for SARS-CoV-2 RNA over the course of the study but remained asymptomatic, with low associated viral RNA copy numbers, no detectable serum IgM and IgG concentrations, and IgA concentrations that decayed rapidly (half-life: 1.3 days). A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees (95% confidence interval [CI] = 3 to 10) would have become infected, with at most 1 of them requiring hospitalization. Our scalable and transferable surveillance plan met its primary objectives and represents a powerful example of an innovative public health initiative dovetailed with scientific discovery. IMPORTANCE The rapid spread of SARS-CoV-2 and the associated COVID-19 has precipitated a global pandemic heavily challenging our social behavior, economy, and health care infrastructure. In the absence of widespread, worldwide access to safe and effective vaccines and therapeutics, public health measures represent a key intervention for curbing the devastating impacts from the pandemic. We are conducting an ongoing clinical study based on frequent, longitudinal workplace disease surveillance to control SARS-CoV-2 transmission among employees and their household members. Our study was successful in surveying the viral and immune response dynamics in two participants with unusual infections: one remained positive for SARS-CoV-2 for 71 days, while the other was asymptomatic, with low associated viral RNA copy numbers. A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees would have become infected, with at most 1 of them requiring hospitalization, underscoring the importance of our program.

Human Vault Nanoparticle Targeted Delivery of Antiretroviral Drugs to Inhibit Human Immunodeficiency Virus Type 1 Infection.

"Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1.

Safety and pharmacokinetics of single, dual, and triple antiretroviral drug formulations delivered by pod-intravaginal rings designed for HIV-1 prevention: A Phase I trial.

BACKGROUND: Intravaginal rings (IVRs) for HIV pre-exposure prophylaxis (PrEP) theoretically overcome some adherence concerns associated with frequent dosing that can occur with oral or vaginal film/gel regimens. An innovative pod-IVR, composed of an elastomer scaffold that can hold up to 10 polymer-coated drug cores (or "pods"), is distinct from other IVR designs as drug release from each pod can be controlled independently. A pod-IVR has been developed for the delivery of tenofovir (TFV) disoproxil fumarate (TDF) in combination with emtricitabine (FTC), as daily oral TDF-FTC is the only Food and Drug Administration (FDA)-approved regimen for HIV PrEP. A triple combination IVR building on this platform and delivering TDF-FTC along with the antiretroviral (ARV) agent maraviroc (MVC) also is under development. METHODOLOGY AND FINDINGS: This pilot Phase I trial conducted between June 23, 2015, and July 15, 2016, evaluated the safety, pharmacokinetics (PKs), and acceptability of pod-IVRs delivering 3 different ARV regimens: 1) TDF only, 2) TDF-FTC, and 3) TDF-FTC-MVC over 7 d. The crossover, open-label portion of the trial (N = 6) consisted of 7 d of continuous TDF pod-IVR use, a wash-out phase, and 7 d of continuous TDF-FTC pod-IVR use. After a 3-mo pause to evaluate safety and PK of the TDF and TDF-FTC pod-IVRs, TDF-FTC-MVC pod-IVRs (N = 6) were evaluated over 7 d of continuous use. Safety was assessed by adverse events (AEs), colposcopy, and culture-independent analysis of the vaginal microbiome (VMB). Drug and drug metabolite concentrations in plasma, cervicovaginal fluids (CVFs), cervicovaginal lavages (CVLs), and vaginal tissue (VT) biopsies were determined via liquid chromatographic-tandem mass spectrometry (LC-MS/MS). Perceptibility and acceptability were assessed by surveys and interviews. Median participant age was as follows: TDF/TDF-FTC group, 26 y (range 24-35 y), 2 White, 2 Hispanic, and 2 African American; TDF-FTC-MVC group, 24.5 y (range 21-41 y), 3 White, 1 Hispanic, and 2 African American. Reported acceptability was high for all 3 products, and pod-IVR use was confirmed by residual drug levels in used IVRs. There were no serious adverse events (SAEs) during the study. There were 26 AEs reported during TDF/TDF-FTC IVR use (itching, discharge, discomfort), with no differences between TDF alone or in combination with FTC observed. In the TDF-FTC-MVC IVR group, there were 12 AEs (itching, discharge, discomfort) during IVR use regardless of attribution to study product. No epithelial disruption/thinning was seen by colposcopy, and no systematic VMB shifts were observed. Median (IQR) tenofovir diphosphate (TFV-DP) tissue concentrations of 303 (277-938) fmol/10(6) cells (TDF), 289 (110-603) fmol/10(6) cells (TDF-FTC), and 302 (177.1-823.8) fmol/10(6) cells (TDF-FTC-MVC) were sustained for 7 d, exceeding theoretical target concentrations for vaginal HIV prevention. The study's main limitations include the small sample size, short duration (7 d versus 28 d), and the lack of FTC triphosphate measurements in VT biopsies. CONCLUSIONS: An innovative pod-IVR delivery device with 3 different formulations delivering different regimens of ARV drugs vaginally appeared to be safe and acceptable and provided drug concentrations in CVFs and tissues exceeding concentrations achieved by highly protective oral dosing, suggesting that efficacy for vaginal HIV PrEP is achievable. These results show that an alternate, more adherence-independent, longer-acting prevention device based on the only FDA-approved PrEP combination regimen can be advanced to safety and efficacy testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431273.

Phase I trial of pod-intravaginal rings delivering antiretroviral agents for HIV-1 prevention: Rectal drug exposure from vaginal dosing with tenofovir disoproxil fumarate, emtricitabine, and maraviroc.

BACKGROUND: Intravaginal rings (IVRs) can deliver antiretroviral (ARV) agents for HIV pre-exposure prophylaxis (PrEP), theoretically overcoming adherence concerns associated with frequent dosing. However, topical vaginal ARV drug delivery has not simultaneously led to sufficient rectal drug exposure to likely protect from HIV infection as a result of receptive anal intercourse (RAI). Unprotected RAI has a higher risk of infection per sex act and, for women, also can be associated with vaginal exposure during a single sexual encounter, especially in higher-risk subsets of women. The physiologically inflamed, activated, immune-cell dense colorectal mucosa is increasingly appreciated as the sexual compartment with highly significant risk; this risk is increased in the setting of co-infections. Ex vivo studies have shown that colorectal tissue and rectal fluid concentrations correlated with HIV protection. Given these important results, efforts to document colorectal compartment ARV drug concentration from pod-IVR delivery was assessed to determine if vaginal application could provide protective ARV levels in both compartments. METHODOLOGY/PRINCIPAL FINDINGS: A crossover clinical trial (N = 6) evaluated 7 d of continuous TDF pod-IVR use, a wash-out phase, followed by 7 d with a TDF-FTC pod-IVR. A subsequent clinical trial (N = 6) consisted of 7 d of continuous TDF-FTC-MVC pod-IVR use. Rectal fluids were collected on Day 7 at IVR removal in all three ARV-exposures (two Phase 1 trials) and drug concentrations quantified by LC-MS/MS. Median rectal fluid concentrations of TFV, the hydrolysis product of the prodrug TDF, were between 0.66 ng mg-1 (TDF pod-IVR group) and 1.11 ng mg-1 (TDF-FTC pod-IVR group), but below the analytical lower limit of quantitation in 5/6 samples in the TDF-FTC-MVC pod-IVR group. Unexpectedly, median FTC (TDF-FTC pod-IVR, 20.3 ng mg-1; TDF-FTC-MVC pod-IVR, 0.18 ng mg-1), and MVC rectal fluid concentrations (0.84 ng mg-1) were quantifiable and higher than their respective in vitro EC50 values in most samples. Due to participant burden in these exploratory trials, rectal fluid was used as a surrogate for rectal tissue, where drug concentrations are expected to be higher. CONCLUSIONS/SIGNIFICANCE: The concentrations of FTC and MVC in rectal fluids obtained in two exploratory clinical trials of IVRs delivering ARV combinations exceeded levels associated with in vitro efficacy in HIV inhibition. Unexpectedly, MVC appeared to depress the distribution of TFV and FTC into the rectal lumen. Here we show that vaginal delivery of ARV combinations may provide adherence and coitally independent dual-compartment protection from HIV infection during both vaginal and receptive anal intercourse.

Highly Attenuated Infection With a Vpr-Deleted Molecular Clone of Human Immunodeficiency Virus-1.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Comparative Assessment of Small and Large Intestine Biopsies for Ex Vivo HIV-1 Pathogenesis Studies.

Ex vivo mucosal explants have become a mainstay of HIV-1 studies using human tissue. In this study, we examine the baseline phenotypic and virologic differences between biopsies derived from the small intestine (SI) and large intestine (LI) for use in ex vivo explant studies. To do this, we collected endoscopic mucosal biopsies from both SI and LI from the same healthy, HIV-seronegative participants. Mucosal mononuclear cell phenotypes and quantity were compared using flow cytometry. Comparative HIV-1 infectibility of the explants was assessed using an ex vivo explant HIV-1 infection assay. We found that all biopsies had similar numbers of T cells per biopsy. While the percentage of CD4+ T cells from SI biopsies expressed significantly more activation markers (CD38, HLA-DR) and HIV coreceptors (CXCR4, CCR5), the absolute numbers of activated CD4+ T cells were similar between both sites. LI explants, however, supported more efficient HIV-1 infection, as evidenced by earlier rise in p24 accumulation and greater percent of infected explants at limiting infectious doses. These results suggest that explants from LI biopsies support more efficient HIV-1 infection than SI biopsies, despite similar numbers of available, activated HIV-1 target cells. These findings highlight important differences in LI and SI explants, which must be considered in designing and interpreting ex vivo HIV-1 infection studies, and suggest that factors within the tissue other than target cell number and activation state may play a role in regulating HIV-1 infection.

Brief Report: Recent Methamphetamine Use Is Associated With Increased Rectal Mucosal Inflammatory Cytokines, Regardless of HIV-1 Serostatus.

BACKGROUND: Methamphetamine use increases the risk of HIV-1 infection among seronegative users and can exacerbate disease progression in HIV-positive users. The biological mechanisms underlying these associations remain unclear. In this cross-sectional pilot study, we examine the associations between recent methamphetamine use and inflammation in the rectal mucosa and peripheral blood compartments in HIV-1 seropositive and seronegative men who have sex with men. METHODS: HIV-seronegative and HIV-seropositive men who have sex with men participants were enrolled (N = 24). Recent methamphetamine use was determined by urine drug screen. Cytokines were quantified using multiplex arrays from collected plasma and rectal sponge samples, and peripheral blood T-cell activation was assessed by flow cytometry. RESULTS: Methamphetamine use was associated with consistently increased rectal inflammatory cytokines, specifically interleukin-6 and tumor necrosis factor-alpha, regardless of HIV-1 serostatus in this pilot study. This association was significant after adjusting for age, HIV-serostatus, and receptive anal intercourse frequency using regression analysis. Similar increases were not uniformly observed in peripheral blood. CONCLUSIONS: Methamphetamine use is associated with increased local mucosal inflammatory cytokine production. These findings may help explain the increased HIV-1 risk seen in methamphetamine users and contribute to increased inflammation among HIV-seropositive users.

Human Semen or Seminal Plasma Does Not Enhance HIV-1BaL Ex Vivo Infection of Human Colonic Explants.

To determine whether human whole semen (WS) and seminal plasma (SP) either previously frozen or freshly acquired altered ex vivo infectibility of human colonic explants or was associated with histology or toxicity changes, which may influence mucosal HIV-1 transmission in vivo. Pooled human semen samples were freshly obtained from study volunteers (never frozen) and from commercial sources (frozen/thawed). Endoscopically acquired rectal biopsies were evaluated for toxicity following titered ex vivo WS/SP exposure by histological grading and by MTT assay. The ex vivo HIV-1 biopsy challenge model was used to evaluate effects of exposure to either previously frozen or freshly acquired WS/SP on HIVBaL infectibility at a range of viral inocula (104-100 TCID50). To evaluate the effects at lower viral inocula of HIV-1 (10-2-102), experiments in the presence or absence of WS/SP were also performed utilizing TZM-bl cells. MTT assays and histological scoring demonstrated no tissue degradation of biopsies when exposed for 2 h to concentrations of 10% or 100% of either fresh or previously frozen WS/SP. Ex vivo biopsy HIV-1 challenge experiments showed no differences in the presence of freshly acquired or previously frozen/thawed WS/SP compared with control; no differences were seen with lower infectious titers on TZM-bl cells. Within the limits of assay sensitivity and variability, these data show no toxicity or significant enhancement of HIV-1 infectibility of human rectal mucosa using the colorectal explant model with either pooled fresh or frozen/thawed nonautologous human semen.

Distinct aging profiles of CD8+ T cells in blood versus gastrointestinal mucosal compartments.

A hallmark of human immunosenescence is the accumulation of late-differentiated memory CD8+ T cells with features of replicative senescence, such as inability to proliferate, absence of CD28 expression, shortened telomeres, loss of telomerase activity, enhanced activation, and increased secretion of inflammatory cytokines. Importantly, oligoclonal expansions of these cells are associated with increased morbidity and mortality risk in elderly humans. Currently, most information on the adaptive immune system is derived from studies using peripheral blood, which contains approximately only 2% of total body lymphocytes. However, most lymphocytes reside in tissues. It is not clear how representative blood changes are of the total immune status. This is especially relevant with regard to the human gastrointestinal tract (GALT), a major reservoir of total body lymphocytes (approximately 60%) and an anatomical region of high antigenic exposure. To assess how peripheral blood T cells relate to those in other locations, we compare CD8+ T cells from peripheral blood and the GALT, specifically rectosigmoid colon, in young/middle age, healthy donors, focusing on phenotypic and functional alterations previously linked to senescence in peripheral blood. Overall, our results indicate that gut CD8+ T cells show profiles suggestive of greater differentiation and activation than those in peripheral blood. Specifically, compared to blood from the same individual, the gut contains significantly greater proportions of CD8+ T cells that are CD45RA- (memory), CD28-, CD45RA-CD28+ (early memory), CD45RA-CD28- (late memory), CD25-, HLA-DR+CD38+ (activated) and Ki-67+ (proliferating); ex vivo CD3+ telomerase activity levels are greater in the gut as well. However, gut CD8+ T cells may not necessarily be more senescent, since they expressed significantly lower levels of CD57 and PD-1 on CD45RO+ memory cells, and had in vitro proliferative dynamics similar to that of blood cells. Compartment-specific age-effects in this cohort were evident as well. Blood cells showed a significant increase with age in proportion of HLA-DR+38+, Ki-67+ and CD25+ CD8+ T cells; and an increase in total CD3+ ex-vivo telomerase activity that approached significance. By contrast, the only age-effect seen in the gut was a significant increase in CD45RA- (memory) and concurrent decrease in CD45RA+CD28+ (naïve) CD8+ T cells. Overall, these results indicate dynamics of peripheral blood immune senescence may not hold true in the gut mucosa, underscoring the importance for further study of this immunologically important tissue in evaluating the human immune system, especially in the context of chronic disease and aging.

Unique microRNA expression in the colonic mucosa during chronic HIV-1 infection.

OBJECTIVE: Chronic HIV-1 infection leads to widespread inflammation and immune dysregulation. The gastrointestinal mucosa, a primary site for HIV-1 replication, is thought to play a significant role in this response. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression, including immune activation and inflammation. Here we investigate miR expression and function in the colonic mucosa during HIV-1 infection. DESIGN AND METHODS: Using miR profiling, we examined miR expression in the colonic mucosa of HIV-infected patients. These miRs were further parsed to identify those that most likely function in HIV-related inflammation. Using bioinformatics tools, we identified potential target genes which were confirmed using in-vitro functional testing. RESULTS: We identified 12 miRs that were differentially expressed in the colonic mucosa of HIV-infected patients with high versus undetectable plasma viral concentrations. Of these, both miR-26a and miR-29a were downregulated in untreated HIV-1 infection, yet not in the colonic mucosa from inflammatory bowel disease. This downregulation occurs within the first hours after infection. These miRs were further shown to directly target IL-6 and STAT3, respectively, with similar changes confirmed in an ex-vivo explant infection model. CONCLUSION: miR-26a and miR-29a levels are decreased in the colonic mucosa during chronic HIV-1 infection, and this change may be initiated during acute infection. Both miRs de-repress the IL-6/STAT3 signaling pathway, which could contribute to increased inflammation during infection. These miRs may represent novel therapeutic targets for HIV-1-associated inflammation in the colonic mucosa.

Highly Human Immunodeficiency Virus-Exposed Seronegative Men Have Lower Mucosal Innate Immune Reactivity.

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes.

The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles. We report here the development of a method for measuring and comparing proliferative capacity of peripheral blood and gut colorectal mucosa-derived CD8 T cells. The protocol involves a 5-day culture of mononuclear leukocyte populations, from blood and gut colorectal mucosa respectively, labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2'-deoxyuridine (BrdU) and stimulated with anti-CD2/3/28-linked microbeads. Variables tested and optimized as part of the protocol development include: mode of T cell stimulation, CFSE concentration, inclusion of a second proliferation marker, BrdU, culture duration, initial culture concentration, and inclusion of autologous irradiated feeder cells. Moving forward, this protocol demonstrates a significant advance in the ability of researchers to study compartment-specific differences of in vitro proliferative dynamics of CD8 T cells, as an indicator of replicative senescence and immunological aging. The study's two main novel contributions are (1) Optimization and adaptation of standard proliferative dynamics blood T cell protocols for T cells within the mucosal immune system. (2) Introduction of the novel technique of combining CFSE and BrdU staining to do so.

Simultaneous Evaluation of Safety, Acceptability, Pericoital Kinetics, and Ex Vivo Pharmacodynamics Comparing Four Rectal Microbicide Vehicle Candidates.

Preexposure prophylaxis (PrEP) of HIV infection with tenofovir-containing regimens is effective, but plagued by poor adherence in some studies. Options for safe, effective, and acceptable PrEP products, especially for men and women at risk of HIV via receptive anal intercourse (RAI), are needed. We performed a randomized, partially blinded, first-in-human evaluation of four candidate rectal microbicide vehicles-aqueous gel, aqueous fluid, lipid gel, and lipid fluid-to select a prototype for further clinical development. Eight seronegative participants received three doses of each product with each dose separated by at least 2 weeks: one dose was given alone without simulated RAI in clinic, another dose was followed by simulated RAI in clinic, and another dose was self-administered at home in the context of RAI with a partner. Assessments included safety, acceptability, colon histology, ex vivo HIV infectivity of colon tissue explants, and colonic luminal distribution of vehicle and HIV surrogates. Adverse events were all mild and mainly sigmoidoscopy associated. There were minor differences in colon distribution of products and little effect of RAI. Vehicle distribution covered 95% (±7% standard deviation) of the distribution of an HIV surrogate in the colonic lumen. The lipid fluid vehicle increased HIV colon tissue infectability 5-fold [log10 p24 0.68 (95% confidence interval 0.08, 1.28)] and aqueous gel provided 6-fold protection [log10 p24 0.80 (95% confidence interval 0.20, 1.41)] compared to no product baseline. Colon permeability of lipid vehicles was more than 10-fold greater than aqueous vehicles. All products received similar acceptability ratings, though trends favored the gel products. Intensive simultaneous assessment of safety and toxicity, luminal and tissue distribution, ex vivo HIV infectivity, and product acceptability in relevant sexual contexts provided clear differentiation among candidate gels very early in product development. We selected the aqueous gel for further development as a rectal microbicide vehicle.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

A Phase 1 Randomized, Open Label, Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Three Formulations of Tenofovir 1% Gel (the CHARM-01 Study).

OBJECTIVES: The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study. METHODS: Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial. RESULTS: All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection. CONCLUSIONS: All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT01575405.

T lymphocyte density and distribution in human colorectal mucosa, and inefficiency of current cell isolation protocols.

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3+ lymphocytes, including CD4+ and CD8+ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r2=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3+ T lymphocytes with 37,400 ± 2,801 cells/mm3 and 33,700 ± 4,324 cell/mm3, respectively. Sigmoid mucosa contained 57% CD3+CD4+ and 40% CD3+CD8+ T lymphocytes which calculates to 21,300 ± 1,476/mm3 and 15,000 ± 275/mm3 T lymphocytes, respectively. Rectal mucosa had 57% CD3+CD4+ and 42% CD3+CD8+ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm3, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm3 and 2,708 ± 245/mm3 CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3+, CD4+ and CD8+ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.