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© 2015 The Authors. Background: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging. Methods: We developed TILDA (Tat/. rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. Findings: In suppressed individuals on ART, we found the median frequency of latently infected CD4. + T cells as estimated by TILDA to be 24. cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p. = 0.011). In untreated HIV disease, the frequency of CD4. + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART. Interpretations: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation. Research in context: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10. ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) is associated with heightened plasma interleukin-10 (IL-10) levels and PD-1 expression. We hypothesized that IL-10 and PD-1 blockade would lead to control of viral rebound following analytical treatment interruption (ATI). Twenty-eight ART-treated, simian immunodeficiency virus (SIV)mac 239 -infected rhesus macaques (RMs) were treated with anti-IL-10, anti-IL-10 plus anti-PD-1 (combo) or vehicle. ART was interrupted 12 weeks after introduction of immunotherapy. Durable control of viral rebound was observed in nine out of ten combo-treated RMs for >24 weeks post-ATI. Induction of inflammatory cytokines, proliferation of effector CD8 + T cells in lymph nodes and reduced expression of BCL-2 in CD4 + T cells pre-ATI predicted control of viral rebound. Twenty-four weeks post-ATI, lower viral load was associated with higher frequencies of memory T cells expressing TCF-1 and of SIV-specific CD4 + and CD8 + T cells in blood and lymph nodes of combo-treated RMs. These results map a path to achieve long-lasting control of HIV and/or SIV following discontinuation of ART., (© 2024. The Author(s).)

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface., Competing Interests: Declaration of interests T.R.-R., K.A.R., T.J.B., J.M.B., C.H.W., D.J.H., K.A.V., E.C.H., O.O.F., and R.C.O. were employed by Merck & Co. during the experimental planning, execution, and preparation of this manuscript. Other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1 + cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4 + T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

In response to infection or immunization, antibodies are produced that provide protection against re-exposure with the same pathogen. These antibodies can persist at high titers for decades and are maintained by bone marrow-resident long-lived plasma cells (LLPC). However, the durability of antibody responses to immunization varies amongst vaccines. It is unknown what factors contribute to the differential longevity of serum antibody responses and whether heterogeneity in LLPC contributes to this phenomenon. While LLPC differentiation has been studied extensively in mice, little is known about this population in humans or non-human primates (NHP). Here, we use multi-omic single-cell profiling to identify and characterize the LLPC compartment in NHP. We identify LLPC biomarkers including the marker CD102 and show that CD102 in combination with CD31 identifies LLPC in NHP bone marrow. Additionally, we find that CD102 is expressed by LLPC in mouse and humans. These results further our understanding of the LLPC compartment in NHP, identify biomarkers of LLPC, and provide tissue-specific single cell references for future studies., (© 2022. Merck & Co., Inc., Rahway, NJ, USA and its affiliates.)

Background: People with HIV (PWH) and community members have advocated for the development of a home-based viral load test device that could make analytical treatment interruptions (ATIs) less burdensome., Objective: We assessed community acceptability of a novel home-based viral load test device., Methods: In 2021, we conducted 15 interviews and 3 virtual focus groups with PWH involved in HIV cure research. We used conventional thematic analysis to analyze the data., Results: PWH viewed the home-based viral load test device as a critical adjunct in ongoing HIV cure trials with ATIs. The ability to test for viral load at home on demand would alleviate anxiety around being off ART. Participants drew parallels with glucometers used for diabetes. A preference was expressed for the home-based test to clearly indicate whether one was detectable or undetectable for HIV to mitigate risk of HIV transmission to partners. Perceived advantages of the device included convenience, sense of control, and no puncturing of veins. Perceived concerns were possible physical marks, user errors and navigating the logistics of mailing samples to a laboratory and receiving test results. Participants expressed mixed effects on stigma, such as helping normalize HIV, but increased potential for inadvertent disclosure of HIV status or ATI participation. Increasing pluri-potency of the device beyond viral load testing (e.g., CD4+ count test) would increase its utility. Participants suggested pairing the device with telemedicine and mobile health technologies., Conclusions: If proven effective, the home-based viral load test device will become a critical adjunct in HIV cure research and HIV care.

Background : HIV cure-directed clinical trials using analytical treatment interruptions (ATIs) require participants to adhere to frequent monitoring visits for viral load tests. Novel viral load monitoring strategies are needed to decrease participant burden during ATIs. Objective : To examine acceptability of a novel home-based blood collection device for viral load testing in the context of two ongoing ATI trials in Philadelphia, PA, United States. Methods : From January 2021 to February 2022, participants completed three in-depth interviews via teleconference during their participation in an ATI: (1) within two weeks of enrollment in the device study, (2) approximately four weeks after beginning to use the device, and (3) within two weeks of the end of the ATI when ART was re-initiated. We used conventional content analysis to analyze the data. Results : We recruited 17 participants: 15 were cisgender males, 1 cisgender female, and 1 transgender woman. We observed an overall 87% success rate in drawing blood with the device from home collection and found overall high acceptance of the device. A mean of 91.5 devices per participant were used for home-based blood collection. Most PWH viewed the device as relatively convenient, painless, easy to use, and a simple solution to frequent blood draws. The main challenge encountered was the inability to completely fill up devices with blood in some cases. Most participants reported positive experiences with mailing blood samples and could see themselves using the device on a regular basis outside of ATIs. Conclusions : Our study showed participant valued the novel home-based peripheral blood collection for viral load testing in the context of ATI trials. More research will be necessary to optimize implementation of the device and to assess whether blood collected can reliably measure viral loads in the context of ATI trials.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor (NNRTI), was approved for treatment of HIV-1 infection in 2018. In the pivotal phase 3 trials, DRIVE-FORWARD and DRIVE-AHEAD, 7 out of 747 (0.9%) treatment-naive participants treated with DOR plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) met protocol-defined virologic failure criteria and showed phenotypic resistance to DOR at week 48. The most common DOR resistance-associated mutation (RAM) detected in 5 of the 7 resistant isolates was F227C. Six isolates bearing NRTI RAMs (M184V and/or K65R) were resistant to lamivudine (3TC) and emtricitabine (FTC) but not to other approved NRTIs. All DOR-resistant isolates were susceptible or hypersusceptible (fold change of <0.25) to islatravir (ISL), a nucleoside reverse transcriptase translocation inhibitor (NRTTI). Isolate hypersusceptibility to ISL required F227C, in contrast to zidovudine, an NRTI, which required M184V. Based on the frequent emergence of F227C, we hypothesized that DOR and ISL would create a combination (DOR/ISL) with a high barrier to resistance. In de novo resistance selection studies in MT4-GFP cells (MT4 cells engineered to express green fluorescent protein), DOR/ISL synergistically prevented viral breakthrough at a threshold of 2× the half-maximal inhibitory concentration (IC 50 ). DOR/ISL exhibited a higher barrier to resistance than DOR/3TC and dolutegravir (DTG)/3TC. Resistance analysis showed no emergence of substitutions at F227, an observation consistent with its ability to confer hypersusceptibility to ISL. Overall, the data demonstrate that DOR/ISL creates a 2-drug combination with a higher barrier to resistance, consistent with the reported clinical activity.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity., Competing Interests: Declaration of interests T.R.R., E.C.H., K.A.V., O.O.F., R.C.O., and D.J.H. are employees of Merck & Co., Inc., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Frequent viral load testing is necessary during analytical treatment interruptions (ATIs) in HIV cure-directed clinical trials, though such may be burdensome and inconvenient to trial participants. We implemented a national, cross-sectional survey in the United States to examine the acceptability of a novel home-based peripheral blood collection device for HIV viral load testing. Between June and August 2021, we distributed an online survey to people with HIV (PWH) and community members, biomedical HIV cure researchers and HIV care providers. We performed descriptive analyses to summarize the results. We received 73 survey responses, with 51 from community members, 12 from biomedical HIV cure researchers and 10 from HIV care providers. Of those, 51 (70%) were cisgender men and 50 (68%) reported living with HIV. Most (>80% overall) indicated that the device would be helpful during ATI trials and they would feel comfortable using it themselves or recommending it to their patients/participants. Of the 50 PWH, 42 (84%) indicated they would use the device if they were participating in an ATI trial and 27 (54%) also expressed a willingness to use the device outside of HIV cure studies. Increasing sensitivity of viral load tests and pluri-potency of the device (CD4 count, chemistries) would augment acceptability. Survey findings provide evidence that viral load home testing would be an important adjunct to ongoing HIV cure-directed trials involving ATIs. Survey findings may help inform successful implementation and uptake of the device in the context of personalized HIV care.

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in the disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae , Ruminococcaceae , and Clostridiaceae . These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies.

We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Covering: up to the end of 2020. The machine learning field can be defined as the study and application of algorithms that perform classification and prediction tasks through pattern recognition instead of explicitly defined rules. Among other areas, machine learning has excelled in natural language processing. As such methods have excelled at understanding written languages (e.g. English), they are also being applied to biological problems to better understand the "genomic language". In this review we focus on recent advances in applying machine learning to natural products and genomics, and how those advances are improving our understanding of natural product biology, chemistry, and drug discovery. We discuss machine learning applications in genome mining (identifying biosynthetic signatures in genomic data), predictions of what structures will be created from those genomic signatures, and the types of activity we might expect from those molecules. We further explore the application of these approaches to data derived from complex microbiomes, with a focus on the human microbiome. We also review challenges in leveraging machine learning approaches in the field, and how the availability of other "omics" data layers provides value. Finally, we provide insights into the challenges associated with interpreting machine learning models and the underlying biology and promises of applying machine learning to natural product drug discovery. We believe that the application of machine learning methods to natural product research is poised to accelerate the identification of new molecular entities that may be used to treat a variety of disease indications.

In the pilot NCT01935089 trial, we tested whether pegylated interferon alpha2b (Peg-IFN-α2b) with antiretroviral therapy (ART) was safe and could impact HIV and immune measures in blood and in gut-associated lymphoid tissue (GALT). Twenty HIV-1 + ART-suppressed individuals received 1 μg/kg/week Peg-IFN-α2b with ART for 20 weeks, with intermediate 4-week analytical ART interruption (ATI). Safety, immune activation, HIV viral load and integrated HIV DNA in blood, and HIV RNA and DNA in gut biopsies were measured. A total of 7/20 participants experienced grade 3-4 adverse events, while 17/20 participants completed the study. Of the 17 participants who completed the study, 8 remained suppressed during ATI, while all 17 were suppressed at end of treatment (EoT). As expected, treatment increased activation of T and natural killer (NK) cells and IFN-stimulated molecule expression on monocytes in periphery. While circulating CD4 + T cells showed a trend for a decrease in integrated HIV DNA, GALT showed a significant decrease in HIV-1 RNA + cells as measured by in situ hybridization along with a reduction in total HIV DNA and cell-associated RNA by EoT. The observed decrease in HIV-1 RNA + cells in GALT was positively associated with the decrease in activated NK cells and macrophages. This study documents for the first time that 20 weeks of immunotherapy with Peg-IFN-α2b+ART (inclusive of a 4-week ATI) is safe and results in an increase in blood and GALT immune activation and in a significant decrease in HIV-1 RNA + cells in GALT in association with changes in innate cell activation.

Emerging evidence in clinical and preclinical studies indicates that success of immunotherapies can be impacted by the state of the microbiome. Understanding the role of the microbiome during immune-targeted interventions could help us understand heterogeneity of treatment success, predict outcomes, and develop additional strategies to improve efficacy. In this review, we discuss key studies that reveal reciprocal interactions between the microbiome, the immune system, and the outcome of immune interventions. We focus on cancer immune checkpoint inhibitor treatment and vaccination as two crucial therapeutic areas with strong potential for immunomodulation by the microbiota. By juxtaposing studies across both therapeutic areas, we highlight three factors prominently involved in microbial immunomodulation: short-chain fatty acids, microbe-associate molecular patterns (MAMPs), and inflammatory cytokines. Continued interrogation of these models and pathways may reveal critical mechanistic synergies between the microbiome and the immune system, resulting in novel approaches designed to influence the efficacy of immune-targeted interventions., Competing Interests: All authors are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and may hold stock in Merck & Co., Inc. Kenilworth, NJ, USA., (Copyright © 2021 Reens, Cabral, Liang, Norton, Therien, Hazuda and Swaminathan.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19., Competing Interests: Competing interest statement: All authors are employees of Merck and Company, Inc. A provisional patent application on the discoveries of this work has been filed., (Copyright © 2021 the Author(s). Published by PNAS.)

Greater than 90% of HIV-1 proviruses are thought to be defective and incapable of viral replication. While replication competent proviruses are of primary concern with respect to disease progression or transmission, studies have shown that even defective proviruses are not silent and can produce viral proteins, which may contribute to inflammation and immune responses. Viral protein expression also has implications for immune-based HIV-1 clearance strategies, which rely on antigen recognition. Thus, sensitive assays aimed at quantifying both replication-competent proviruses and defective, yet translationally competent proviruses are needed to understand the contribution of viral protein to HIV-1 pathogenesis and determine the effectiveness of HIV-1 cure interventions. Previously, we reported a modified HIV-1 gag p24 digital enzyme-linked immunosorbent assay with single molecule array (Simoa) detection of cell-associated viral protein. Here we report a novel p24 protein enrichment method coupled with the digital immunoassay to further extend the sensitivity and specificity of viral protein detection. Immunocapture of HIV gag p24 followed by elution in a Simoa-compatible format resulted in higher protein recovery and lower background from various biological matrices and sample volumes. Quantification of as little as 1 fg of p24 protein from cell lysates from cells isolated from peripheral blood or tissues from ART-suppressed HIV participants, as well as simian-human immunodeficiency virus-infected non-human primates (NHPs), with high recovery and reproducibility is demonstrated here. The application of these enhanced methods to patient-derived samples has potential to further the study of the persistent HIV state and examine in vitro response to therapies, as well as ex vivo study of translationally competent cells from a variety of donors., Competing Interests: GW, CC, QH, DH, BH, and PZ were employed by Merck & Co., Inc., (Copyright © 2021 Wu, Cheney, Huang, Hazuda, Howell and Zuck.)

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture ® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts., Competing Interests: All authors are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and may own stock or hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that has led to a global economic disruption and collapse. With several ongoing efforts to develop vaccines and treatments for COVID-19, understanding the molecular interaction between the coronavirus, host cells, and the immune system is critical for effective therapeutic interventions. Greater insight into these mechanisms will require the contribution and combination of multiple scientific disciplines including the techniques and strategies that have been successfully deployed by chemical biology to tease apart complex biological pathways. We highlight in this review well-established strategies and methods to study coronavirus-host biophysical interactions and discuss the impact chemical biology will have on understanding these interactions at the molecular level., Competing Interests: E. C. H., K. A. V., D. J. H., R. C. O., and O. O. F. are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA., (This journal is © The Royal Society of Chemistry.)

Background: Doravirine (DOR) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) approved for the treatment of HIV-1 infection in patients with no known DOR resistance-associated mutations. DOR was rationally designed to address limitations associated with other approved NNRTIs, particularly resistance from common NNRTI resistance-associated mutants containing K103N, Y181C, or G190A reverse transcriptase substitutions., Setting: Data to date from both in vitro studies and clinical trials have been compiled to summarize the resistance profile of DOR., Methods: We analyzed data from in vitro studies and phase 2 and 3 trials to assess the emergence of resistance-associated mutations and their impact on efficacy among participants treated with DOR., Results: DOR exhibited a distinct resistance profile compared with efavirenz and rilpivirine in vitro and in vivo; mutant viruses that were resistant to DOR showed limited cross-resistance to efavirenz and rilpivirine. In clinical trials, the development of DOR resistance-associated substitutions in reverse transcriptase was uncommon., Conclusion: Overall, minimal cross-resistance across NNRTIs was observed for DOR and limited development of DOR-related resistance. These data should assist clinicians in further understanding the resistance profile of DOR, so appropriate treatment decisions can be made for their patients.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Histone deacetylase inhibitors (HDACi) are the most widely studied HIV latency-reversing agents (LRAs). The HDACi suberoylanilide hydroxamic acid (vorinostat [VOR]) has been employed in several clinical HIV latency reversal studies, as well as in vitro models of HIV latency, and has been shown to effectively induce HIV RNA and protein expression. Despite these findings, response to HDACi can vary, particularly with intermittent dosing, and information is lacking on the relationship between the host transcriptional response and HIV latency reversal. Here, we report on global gene expression responses to VOR and examine the longevity of the transcriptional response in various cellular models. We found that many genes are modulated at 6 h post-VOR treatment in HCT116, Jurkat, and primary resting CD4 T cells, yet return to baseline levels after an 18-h VOR-free period. With repeat exposure to VOR in resting CD4 T cells, we found similar and consistent transcriptional changes at 6 h following each serial treatment. In addition, serial exposure in HIV-infected suppressed donor CD4 T cells showed consistent transcriptional changes after each exposure to VOR. We identified five host genes that were strongly and consistently modulated following histone deacetylase (HDAC) inhibition; three (H1F0, IRGM, and WIPI49) were upregulated, and two (PHF15 and PRDM10) were downregulated. These genes demonstrated consistent modulation in peripheral blood mononuclear cell (PBMC) samples from HIV-positive (HIV + ) participants who received either single or multiple doses of 400 mg of VOR. Interestingly, the host transcriptional response did not predict induction of cell-associated HIV RNA, suggesting that other cellular factors play key roles in HIV latency reversal in vivo despite robust HDACi pharmacological activity. IMPORTANCE Histone deacetylase inhibitors are widely studied HIV latency-reversing agents (LRAs). VOR, an HDACi, induces histone acetylation and chromatin remodeling and modulates host and HIV gene expression. However, the relationship between these events is poorly defined, and clinical studies suggest diminished HIV reactivation in resting CD4 T cells with daily exposure to VOR. Our study provides evidence that VOR induces a consistent level of host cell gene transcription following intermittent exposure. In addition, in response to VOR exposure a gene signature that was conserved across single and serial exposures both in vitro and in vivo was identified, indicating that VOR can consistently and reproducibly modulate transcriptional host responses. However, as the HIV response to HDACi declines over time, other factors modulate viral reactivation in vivo despite robust HDAC activity. The identified host gene VOR biomarkers can be used for monitoring the pharmacodynamic activity of HDAC inhibitors., (Copyright © 2020 Maxwell et al.)

Background: MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is a novel reverse transcriptase-translocation inhibitor., Methods: We assessed MK-8591 as preexposure prophylaxis in the rhesus macaque model of intrarectal challenge with simian/human immunodeficiency virus (SHIV). In study 1, 8 rhesus macaques received 3.9 mg/kg of MK-8591 orally on day 0 and once weekly for the next 14 weeks. Eight controls were treated with vehicle. All rhesus macaques were challenged with SHIV109CP3 on day 6 and weekly for up to 12 challenges or until infection was confirmed. The dose of MK-8591 was reduced to 1.3 and 0.43 mg/kg/week in study 2 and further to 0.1 and 0.025 mg/kg/week in study 3. In studies 2 and 3, each dose was given up to 6 times once weekly, and animals were challenged 4 times once weekly with SHIV109CP3., Results: Control macaques were infected after a median of 1 challenge (range, 1-4 challenges). All treated animals in studies 1 and 2 were protected, consistent with a 41.5-fold lower risk of infection (P < .0001, by the log-rank test). In study 3, at a 0.1-mg/kg dose, 2 rhesus macaques became infected, consistent with a 7.2-fold lower risk of infection (P = .0003, by the log-rank test). The 0.025-mg/kg dose offered no protection., Conclusions: These data support MK-8591's potential as a preexposure prophylaxis agent., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Natural products represent a rich reservoir of small molecule drug candidates utilized as antimicrobial drugs, anticancer therapies, and immunomodulatory agents. These molecules are microbial secondary metabolites synthesized by co-localized genes termed Biosynthetic Gene Clusters (BGCs). The increase in full microbial genomes and similar resources has led to development of BGC prediction algorithms, although their precision and ability to identify novel BGC classes could be improved. Here we present a deep learning strategy (DeepBGC) that offers reduced false positive rates in BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing machine-learning tools. We supplemented this with random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable putative BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a major addition to in-silico BGC identification., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

HIV persists in latently infected CD4 + T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4 + T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption.

Objectives: The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles., Methods: Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells. RT variants were purified via avidin affinity columns., Results: Compound A displayed equal or greater potency against many common NNRTI-resistant RTs (K103N and Y181C RTs) relative to WT RT. Despite possessing certain NNRTI-like properties, such as being unable to inhibit an engineered variant of RT lacking an NNRTI-binding pocket, we found that compound A was dependent on Mg2+ for binding to RT. Optimization of compound A led to more potent analogues, which retained similar activities against WT and K103N mutant viruses with submicromolar potency in a cell-based assay. One of the analogues, compound G, was crystallized in complex with RT and the structure was determined at 2.6 Å resolution. The structure indicated that compound G simultaneously interacts with the active site (Asp186), the highly conserved primer grip region (Leu234 and Trp229) and the NNRTI-binding pocket (Tyr188)., Conclusions: These findings reveal a novel class of RT bifunctional inhibitors that are not sensitive to the most common RT mutations, which can be further developed to address the deficiency of current RT inhibitors., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3 bispecific antibody. These findings extend beyond classical nucleic acid endpoints, which are confounded by the predominance of mutated, defective proviruses and, of paramount importance, enable assessment of cells making HIV protein that can now be targeted by immunological approaches.

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection., Competing Interests: CONFLICTS OF INTEREST The authors have nothing else to disclose.

HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π-π stacking plays a crucial role in the binding of NNRTIs to RT., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Research toward a cure for human immunodeficiency virus type 1 (HIV-1) infection has joined prevention and treatment efforts in the global public health agenda. A major approach to HIV eradication envisions antiretroviral suppression, paired with targeted therapies to enforce the expression of viral antigen from quiescent HIV-1 genomes, and immunotherapies to clear latent infection. These strategies are targeted to lead to viral eradication--a cure for AIDS. Paired testing of latency reversal and clearance strategies has begun, but additional obstacles to HIV eradication may emerge. Nevertheless, there is reason for optimism that advances in long-acting antiretroviral therapy and HIV prevention strategies will contribute to efforts in HIV cure research and that the implementation of these efforts will synergize to markedly blunt the effect of the HIV pandemic on society., (Copyright © 2016, American Association for the Advancement of Science.)

Objectives: Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission., Methods: Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD-scid-gamma) and humanized BLT (bone marrow-liver-thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice., Results: Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration., Conclusions: These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The latent reservoir in resting CD4(+) T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents., Results: Here, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4(+) T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4(+) T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells., Conclusion: We have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Doravirine (DOR), which is currently in a phase 3 clinical trial, is a novel human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI). DOR exhibits potent antiviral activity against wild-type virus and K103N, Y181C, and K103N/Y181C mutant viruses, with 50% inhibitory concentrations (IC50s) of 12, 21, 31, and 33 nM, respectively, when measured in 100% normal human serum (NHS). To assess the potential for DOR to suppress NNRTI-associated and rilpivirine (RPV)-specific mutants at concentrations achieved in the clinic setting, inhibitory quotients (IQs) were calculated by determining the ratio of the clinical trough concentration over the antiviral IC50for each virus with DOR and RPV and efavirenz (EFV). DOR displayed IQs of 39, 27, and 25 against the K103N, Y181C, and K103N/Y181C mutants, respectively. In contrast, RPV exhibited IQs of 4.6, 1.4, and 0.8, and EFV showed IQs of 2.5, 60, and 1.9 against these viruses, respectively. DOR also displayed higher IQs than those of RPV and EFV against other prevalent NNRTI-associated mutants, with the exception of Y188L. Both DOR and EFV exhibited higher IQs than RPV when analyzed with RPV-associated mutants. Resistance selections were conducted with K103N, Y181C, G190A, and K103N/Y181C mutants at clinically relevant concentrations of DOR, RPV, and EFV. No viral breakthrough was observed with DOR, whereas breakthrough viruses were readily detected with RPV and EFV against Y181C and K103N viruses, respectively. These data suggest that DOR should impose a higher barrier to the development of resistance than RPV and EFV at the concentrations achieved in the clinic setting., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasma exposures of the parent molecules.

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Doravirine (DOR, formerly known as MK-1439) is a human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is currently in phase 2b clinical trials. In vitro resistance selection of subtype B virus (MT4-green fluorescent protein [GFP] cells), as well as subtype A and C viruses (MT4-GFP/CCR5 cells) was conducted with DOR, rilpivirine (RPV), and efavirine (EFV) under low-multiplicity-of-infection conditions in a 96-well format. Resistance selection was performed with escalating concentrations of the NNRTIs ranging from the 95% effective concentration (1 × EC(95)) to 1,000 × EC(95) in the presence of 10% fetal bovine serum. In the resistance selection of subtype B virus with DOR, a V106A mutant virus led to two mutation pathways, followed by the emergence separately of either F227L or L234I. In the resistance selection of subtype A and C viruses, similar mutation development pathways were detected, in which a V106A or V106M mutant was also the starting virus in the pathways. Mutations that are commonly associated with RPV and EFV in clinical settings were also identified in subtype B viruses such as the E138K and K103N mutants, respectively, in this in vitro resistance selection study. The susceptibility of subtype B mutant viruses selected by DOR, RPV, and EFV to NNRTIs was evaluated. Results suggest that mutant viruses selected by DOR are susceptible to RPV and EFV and mutants selected by RPV and EFV are susceptible to DOR. When the replication capacity of the V106A mutant was compared with that of the wild-type (WT) virus, the mutant virus was 4-fold less fit than the WT virus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)