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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.

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.

Since the approval of nevirapine, the first HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) in 1996, NNRTIs have helped play a critical role in maintaining viral suppression in people living with HIV. The many positive attributes of the class, including potency and long plasma half-life, make them attractive drug discovery targets. Given the availability of multiple once-daily integrase-based treatments for HIV-1 infection, the challenge to develop a new antiretroviral agent that addresses the needs of today's patients is formidable. However, with the increased availability of antiretrovirals for treatment and new pre-exposure prophylaxis guidelines, which should globally expand the use of antiretrovirals in prevention, it will be increasingly important to have access to multiple regimens with options from different classes that are well tolerated and convenient to ensure a sustained impact on the global epidemic. Many attempts to improve upon the NNRTI class have failed to deliver a desirable clinical profile consistent with the current landscape of treatment options. Doravirine is the only NNRTI to successfully advance through phase 3 clinical development and approval in recent years. Learning from the liabilities of approved NNRTIs, as well as past development failures, facilitated a rational approach to the discovery of doravirine by focusing on addressing the known safety/tolerability issues of commonly prescribed NNRTIs, such as central nervous system toxicity with efavirenz and potential cardiotoxicity due to off-target effects on cardiac ion channels with rilpivirine, using structural biology and characterization of resistance in vitro to address resistance liabilities and concentrating on the metabolic profile to limit the potential for drug-drug interactions. These preclinical efforts were critical to the design and selection of doravirine as a novel NNRTI that possessed the desired next-generation profile with the ultimate proof that these attributes translate to patients derived from clinical trials.

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., Patient drug regime compliance is a major issue; sustained release implants could address this. Here, the authors report on a phase inverted in situ forming implant of PLGA for the sustained release of antiretroviral drugs and optimize and demonstrate the release of 6 different drugs over a period of up to a year.

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 more accurate BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing tools. We supplemented this with downstream random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a significant step forward forin-silicoBGC identification.

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.

The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC 90 ) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC 90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.

The persistence of HIV reservoirs, including latently infected, resting CD4(+) T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32(+) CD4(+) T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4(+) T cells in peripheral blood or lymphoid tissue; isolated CD32(+) resting CD4(+) T cells accounted for less than 3% of the total HIV DNA in CD4(+) T cells. Cell-associated HIV DNA and RNA loads in CD4(+) T cells positively correlated with the frequency of CD32(+) CD69(+)CD4(+)T cells but not with CD32 expression on resting CD4(+) T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV- infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4(+) T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4(+) T cells enriched for transcriptionally active HIV after long-term ART.

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.

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 A 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.

Three integrase strand transfer inhibitors have now been approved for the treatment of HIV infection, raltegravir, cobicistat-boosted elvitegravir, and dolutegravir. Each of these agents selects for unique signature mutations; however, there can be significant cross resistance among all three drugs when multiple mutations are present or are presented in the context of different genetic backgrounds such as non B-subtypes. Many of the mutations that are associated with integrase inhibitor resistance have a profound effect on integrase function and viral replication and thus, while only one or two mutations may be sufficient to impact susceptibility, virologic failure and treatment-associated resistance have been infrequent with all three drugs to date.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC 50 ) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC 95 ) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC 95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

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.

Background Vaniprevir with P/R improved SVR rates over P/R alone in treatment-experienced patients with chronic HCV-genotype 1 infection, but treatment failure presents therapeutic challenges. We identified RAVs from non-cirrhotic patients failing to achieve SVR on vaniprevir-containing regimens from a dose/duration-ranging trial of triple-combination therapy. Methods Using population analysis, resistance sequencing was performed on all baseline samples and on samples at virologic failure in the vaniprevir arms. Longitudinal clonal analyses were performed on viral isolates from six vaniprevir recipients experiencing breakthrough viremia. Results Baseline RAVs were detected in two patients subsequently experiencing virologic failure. At virologic failure, the majority of RAVs had substitutions at R155, A156, or D168. Clonal analyses identified novel double/triple variants emerging with continuing vaniprevir dosing. Conclusions RAVs were predominantly observed at R155, A156, and/or D168 during virologic failure on vaniprevir/P/R. Double/triple RAVs were identified in patients remaining viremic on triple therapy, suggesting evolution of resistance under selective pressure.

The identification of hepatitis C virus (HCV) as the causative agent of non-A and non-B hepatitis, over 20 years ago, fueled an intensive effort to develop direct-acting antivirals targeting the viral polymerase and protease, two key proteins critical for HCV replication. However, it took more than two decades for these efforts to be realized with boceprevir, one of the two HCV protease inhibitors approved for treatment of HCV infection in 2011. The development of boceprevir is a major advancement in the ability to treat HCV infection and a significant step toward the long-term goal of eradicating chronic HCV infection. Both as a first-in-class agent and an entirely new modality for treating HCV infection, many challenges were encountered during the discovery and development of this compound. The lessons learned in overcoming these obstacles offer insights and pave the way for the newly emerging field of HCV antiviral therapeutics. This paper will describe the discovery and development of a first-in-class direct antiviral treatment for chronic hepatitis C infection, boceprevir, marketed around the world as VictrelisTM.

BACKGROUND Resistance to direct-acting antivirals represents a new challenge in the treatment of chronic hepatitis C. METHODS SPRINT-1 was a randomized study of treatment-naive patients with genotype (G) 1 hepatitis C infection (n=595) that evaluated the safety and efficacy of boceprevir (BOC) when added to pegylated interferon-α2b plus ribavirin (PR). Plasma samples collected at protocol-specified visits were analysed by population sequencing for detection of BOC-associated resistance-associated variants (RAVs). RESULTS A total of 17/24 (71%) patients randomized to BOC with baseline RAVs achieved sustained virological response (SVR). V55A/I (n=14), Q41H (n=11) and T54S (n=9) were the most frequently detected polymorphisms at baseline. Seven non-SVR patients with baseline RAVs had V55A (relapse, n=3; breakthrough, n=1; and non-response, n=1) and/or R155K (non-response, n=2). In total, 63/144 (44%) patients with sequenced post-baseline samples (2 SVR, 61 non-SVR) had detectable RAVs after BOC treatment (G1a: R155K [39/49; 80%], V36M [37/49; 76%] and T54S [24/49; 49%]; G1b: T54S [3/11; 27%], T54A [4/11; 35%], A156S [2/11; 18%] and V170A [2/11; 18%]). RAV frequency varied according to the virological response: 90%, 67%, 27% and 37% of breakthrough, incomplete virological response, relapse and non-responder patients, respectively, had post-baseline RAVs present. Similar RAVs were identified in both the PR lead-in and no-lead-in arms and the frequency of post-baseline RAVs was highest in the low-dose ribavirin arm. CONCLUSIONS SVR rates were not compromised among patients with RAVs at baseline; however, a lower starting mg/kg dose of ribavirin was associated with a higher frequency of post-baseline RAVs.

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.

Design The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. Methods Human CD4+ T cells were treated ex vivo with Phorbol 12-myristate 13-acetate, prostatin or ingenol-3-angelate. At 3 h and 24 h post-treatment, cells were harvested and RNA-Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT-PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. Results Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. Conclusion Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation.

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.

Background: Use of direct-acting antiviral drugs (DAAs) that target the Hepatitis C virus may be hampered by the rapid selection of viral strains that harbour drug resistance-associated variants (RAVs). These RAVs are often associated with a fitness cost and tend to occur on low frequency strains within treatment naive subjects. To address the clinical relevance of low frequency RAVs in the setting of DAAs, this study utilised a Primer ID ultra-deep sequencing approach to mitigate PCR errors and bias to accurately quantify viral sequences in subjects that failed DAA treatment. Methods: Subjects were enrolled in the follow-up study P05063 of previous treatment with boceprevir and all had detectable RAVs at virological failure (VF) based on Sanger-based population sequencing. Twelve subjects had three time-points available: baseline, VF, and follow-up (median 830.5 days). Viral RNA was amplified using unique primer identifiers (primer IDs) and sequenced using 454 ultra-deep sequencing. Results: The sequencing strategy used in this study improved the detection of clinically relevant low frequency strains bearing RAVs compared to population sequencing and showed that these strains can persist for up to two years post-treatment failure. Strains carrying multiple RAVs were common in breakthrough viruses. Putative compensatory mutations were identified. Conclusions: The Primer ID ultra-deep sequencing approach identifies RAVs that can reduce drug sensitivity at levels below the detection threshold for population sequencing. The approach also removes PCR errors and biases, suggesting this sequencing strategy should become the standard approach by which to perform temporal quasispecies studies and resistance screening.

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.

BACKGROUND A central challenge to emerging efforts to cure HIV infection is the persistence of quiescent but replication-competent proviral genomes in resting CD4 + T lymphocytes and, to an unknown extent, in other cell populations. Targeted approaches are sought to reverse latency, induce viral antigen expression within formerly latently infected cells, and use immune clearance mechanisms to eradicate persistent infection. Small-molecule HIV latency-reversing agents (LRAs) capable of modulating pathways that control HIV-1 latency are the first tools to be studied in this effort. However, despite the successful reversal of latency, the only clinical interventions to date producing a significant decrease in the viral reservoir involved bone marrow transplantation. Moreover, whether the HIV-1 RNA expression induced by LRAs leads to durable viral protein presentation on the surface of infected cells sufficient to allow immune mediated clearance is unknown. Because multiple mechanisms are involved in maintaining the transcriptional silence of HIV-1, a combination of LRAs may be necessary to effectively perturb persistent HIV-1 infection. Defining the cells that harbor persistent HIV-1 is technically challenging, burdening the development of antilatency therapy. Last, immune interventions designed to clear persistently infected cells have yet to be combined with LRAs in a successful, coordinated therapeutic strategy. ADVANCES Histone deacetylase (HDAC) inhibitors have been the most widely investigated LRAs, inducing cell-associated HIV-1 RNA in four clinical studies. The complex nature of the mechanisms that restrict HIV-1 expression of the population of latent integrated proviruses suggests the hypothesis that combinations of agents could more effectively disrupt latency. Numerous in vitro studies support this hypothesis, but thus far, only one animal model study used several LRAs; although results appeared promising, the effect of this combination was not directly assessed. A study of latently infected cells obtained from HIV-1 + donors on antiretroviral therapy revealed that instantaneous reversal of latency across all cells harboring integrated proviruses may be challenging and suggests that effective LRA strategies will likely need to be delivered serially over time. This study also illustrates the limitations of assessing the persistence of latent infection via HIV-1 RNA expression because a majority of such RNA transcripts are defective and therefore irrelevant. New approaches,such as novel techniques that can quantitate the frequency of rare cells in a large population that are capable of expressing viral antigen, may surmount this challenge. Other recent efforts have examined the obstacles to clearance of persistent, latent infection. For example, the latent reservoir harbors viral mutant subspecies that have previously escaped the extant antiviral immune response. Tissue sanctuaries that may be poorly accessed by the cytotoxic T lymphocyte (CTL) response have been described, as has the potential for some LRAs to interfere with CTL activity. Whether the extent of antigen presentation induced by the current generation of LRAs is sufficient to allow targeting and clearance of infected cells remains unknown. OUTLOOK Although the challenges of latent HIV-1 infection are daunting, the stability of the latent pool over years of antiviral therapy gives hope that an effective perturbation of the homeostasis that maintains the latent pool may allow substantial depletion, and eventually eradication, of persistent infection. The steady advances in animal models of HIV-1 latency, tools for the assessment of persistent infection, LRAs to disrupt latency, and emerging immunotherapeutics to clear persistently infected cells suggest that the first effective combination studies may be close at hand. Such studies will likely represent progress toward the goal of HIV-1 cure while also revealing new challenges to be overcome.

Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection [1]. Inducing the expression of latent genomes within resting CD4+ T cells is the primary strategy to clear this reservoir [2]. While histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA or vorinostat, VOR) can disrupt HIV-1 latency in vitro [3–5], the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Therefore we isolated the circulating resting CD4+ T cells of patients in whom viremia was fully suppressed by antiretroviral therapy (ART), and directly studied the effect of VOR in this latent reservoir. In each of eight patients studied, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4+ cells (mean increase 4.8-fold). This is the first demonstration that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in man, provides proof-of-concept for HDAC inhibitors as a new therapeutic class, and defines a precise approach to test novel strategies to directly attack and eradicate latent HIV infection.

MK-6186 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) which displays subnanomolar potency against wild-type (WT) virus and the two most prevalent NNRTI-resistant RT mutants (K103N and Y181C) in biochemical assays. In addition, it showed excellent antiviral potency against K103N and Y181C mutant viruses, with fold changes (FCs) of less than 2 and 5, respectively. When a panel of 12 common NNRTI-associated mutant viruses was tested with MK-6186, only 2 relatively rare mutants (Y188L and V106I/Y188L) were highly resistant, with FCs of >100, and the remaining viruses showed FCs of 10, whereas only 29% of the mutant viruses displayed greater than 10-fold resistance to MK-6186. To determine whether MK-6186 selects for novel resistance mutations, in vitro resistance selections were conducted with one isolate each from subtypes A, B, and C under low-multiplicity-of-infection (MOI) conditions. The results showed a unique mutation development pattern in which L234I was the first mutation to emerge in the majority of the experiments. In resistance selection under high-MOI conditions with subtype B virus, V106A was the dominant mutation detected in the breakthrough viruses. More importantly, mutant viruses selected by MK-6186 showed FCs of

Raltegravir is the first integrase strand transfer inhibitor approved for the treatment of HIV-1 infection. As the first agent in this new class of antiretroviral therapies, raltegravir has demonstrated safety and efficacy in treatment-naive as well as heavily pretreated HIV-infected patients failing therapy with multidrug-resistant virus. Raltegravir has a favorable drug interaction profile that permits both administration to a wide, demographically diverse patient population and coadministration with many other therapeutic agents, including antiretroviral agents and supportive medications, without restrictions or dose adjustment. Data through 96 weeks of follow-up in three phase III studies, protocol 021 (STARTMRK) in treatment-naive patients, and protocols 018 (BENCHMRK-1) and 019 (BENCHMRK-2) in treatment-experienced patients, demonstrated the potent and durable antiretroviral and immunologic effects and the favorable long-term safety profile of raltegravir in both treatment-naive and treatment-experienced patients. Raltegravir represents an important addition to the current armamentarium for the treatment of HIV infection.

Raltegravir is highly efficacious in the treatment of HIV-1 infection. The prevalence and impact on virologic outcome of low-frequency resistant mutations among HIV-1-infected patients not previously treated with raltegravir have not been fully established. Samples from HIV treatment-experienced patients entering a clinical trial of raltegravir treatment were analyzed using a parallel allele-specific sequencing (PASS) assay that assessed six primary and six secondary integrase mutations. Patients who achieved and sustained virologic suppression (success patients, n = 36) and those who experienced virologic rebound (failure patients, n = 35) were compared. Patients who experienced treatment failure had twice as many raltegravir-associated resistance mutations prior to initiating treatment as those who achieved sustained virologic success, but the difference was not statistically significant. The frequency of nearly all detected resistance mutations was less than 1% of viral population, and the frequencies of mutations between the success and failure groups were similar. Expansion of pre-existing mutations (one primary and five secondary) was observed in 16 treatment failure patients in whom minority resistant mutations were detected at baseline, suggesting that they might play a role in the development of drug resistance. Two or more mutations were found in 13 patients (18.3%), but multiple mutations were not present in any single viral genome by linkage analysis. Our study demonstrates that low-frequency primary RAL-resistant mutations were uncommon, while minority secondary RAL-resistant mutations were more frequently detected in patients naïve to raltegravir. Additional studies in larger populations are warranted to fully understand the clinical implications of these mutations.

Background First-generation NNRTIs with nucleoside reverse transcriptase inhibitors are effective in sustaining HIV-1 suppression but development of resistant mutants is often seen in patients whose regimens fail. These NNRTIs are also associated with safety/tolerability issues, such as CNS and rash. Despite intensive efforts in developing NNRTIs with improved resistance and safety profiles, only two next-generation NNRTIs were successfully developed over the last decade, etravirine (ETR) and rilpivirine (RPV). RPV is less efficacious in patients with high viral load and ETR is only approved for treating experienced patients. Lessons from the limitations of approved NNRTIs and past development failures informed a rational approach to the development of DOR. Methods This review describes the development of DOR, which applied resistance selection and crystallography studies to improve resistance profiles, qEEG studies to evaluate CNS effects, and animal studies to optimize pharmacokinetic profiles, with confirmation in clinical trials. Results DOR demonstrated potent in vitro activity against wild-type virus and mutant viruses containing common NNRTI resistance mutations (K103N, Y181C, G190A, E138K, and K103N/Y181C), and selection studies suggested a unique resistance profile characterized by the emergence of a mutation at position 106 (V106A/M) with additional substitutions, such as F227C, required for high-level resistance. Related analogs were devoid of qEEG effects in rats and nonhuman primates. The metabolic profile was devoid of induction potential, suggesting a benign drug interaction profile. In the ongoing clinical studies, resistance rates were lower than first-generation NNRTIs, with no clinically meaningful drug interactions, and DOR has been generally well tolerated with favorable safety, neuropsychiatric, and lipid profiles. Conclusion Current clinical experience confirmed the preclinical profile of DOR. DOR is a unique NNRTI, distinguished by its low risk of resistance and excellent tolerability. DOR demonstrated a superior neuropsychiatric profile compared with EFV, a superior lipid profile vs. DRV+r and EFV, and a favorable drug–drug interaction profile comparable to integrase strand transfer inhibitors. Disclosures C. Hwang, Merck & Co., Inc.: Employee and Shareholder, Salary. M. T. Lai, Merck & Co., Inc.: Employee and Shareholder, Salary. D. Hazuda, Merck & Co., Inc.: Employee and Shareholder, Salary.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.

The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.

Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4 + T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4 + T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4 + T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

The early events of HIV-1 replication are highlighted by reverse transcription and integration, and the reverse transcriptase and integrase enzymes are important therapeutic targets. Integration proceeds through a series of steps including assembly of integrase on the viral donor DNA ends, 3′-processing, and DNA strand transfer. First generation integrase assays typically included all biochemical reagents in solution where excess donor substrate could serve as the target for DNA strand transfer. These conditions, though valuable for understanding mechanistic aspects of HIV-1 integration, fell short of critical pharmacological designs as most early inhibitors were found to block assembly instead of enzyme function. Second generation designs, which decoupled assembly from DNA strand transfer, afforded the specificity required to identify clinically relevant compounds. Here, we describe versatile scintillation proximity-based assays whereby integrase is assembled onto donor DNA that is immobilized onto the surface of beads. Immobilization and subsequent washing of excess donor DNA eliminates its potential to serve as target DNA, allowing investigation of the DNA strand transfer reaction in isolation. Assembled complexes can be used in high-throughput DNA strand transfer assays if radio labeled target DNA is employed or in integrase binding assays using a suitable radioligand.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2′- C -methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC 50 ) of 0.3 μM (EC 90 = 1.3 μM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log 10 IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log 10 , and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

The first integrase inhibitor licensed to treat HIV-1 infection was approved in late 2007, more than a decade after the introduction of the first inhibitors of the HIV-1 reverse transcriptase and protease. The unique biochemical and molecular mechanism of action of this novel class of antiretroviral drugs is the fundamental basis for their activity in treating multidrug-resistant HIV-1 infection and is important for understanding both the cellular and in vivo pharmacology and metabolism of these agents. In addition, available pharmacokinetic and drug interaction data for raltegravir and elvitegravir, the two integrase inhibitors that are the most advanced in clinical development to date, are reviewed.

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p < 0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Reverse transcriptase (RT) plays an essential role in the HIV-1 replication process, which converts a single-strand RNA into a double-strand DNA via polymerase and RNase H activities. Therefore, inhibition of RT has been one of the primary therapeutic strategies for suppressing the replication of HIV-1. To facilitate the process of discovering the next generation of antiretroviral agents, this study presents a highly sensitive and nonradioactive RT polymerase assay that is based on electrochemiluminescence (ECL) technology, where a ruthenylated dUTP (Ru-dUTP) is employed as one of the dNTPs. The concentration of the RT enzymes required for the assay can be as low as 1 pM, enabling us to evaluate inhibitors with low picomolar potency. More importantly, the assay is capable of detecting endogenous RT activity in cell-free viruses. Therefore, the assay was applied to monitor the development of resistance mutation(s) by viruses under the treatment with a non-nucleoside reverse transcriptase inhibitor (NNRTI) in cell culture. The magnitude of resistance of the resulting mutant viruses was assessed directly by the assay, eliminating the need for cloning, expressing, and purifying the RT mutants.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

Raltegravir (MK-0518) belongs to the new class of HIV integrase inhibitors. To date, there have been no reports investigating the potential for differential effects on viral dynamics with integrase inhibitors relative to current antiretroviral drugs.Patients in this phase II study (P004) were antiretroviral treatment naive. Part 1 of this study compared monotherapy with raltegravir (100 mg, 200 mg, 400 mg, or 600 mg twice daily) with placebo over 10 days. In part 2, patients were enrolled for 48 weeks of combination therapy, with randomization to one of the four dosages of raltegravir or to efavirenz, in addition to tenofovir and lamivudine. Mathematical models were used to investigate processes underlying viral dynamics.From day 15 through to day 57, individuals in the raltegravir arm were significantly more likely to have HIV RNA50 copies/ml (Por = 0.047). Plasma viral loads were 70% lower at initiation of second-phase decay for individuals taking raltegravir than for those taking efavirenz (P0.0001). This challenges the current hypothesis that second-phase virus originates from infected long-lived cells, as an integrase inhibitor should not impact on viral production from this cell population. Mathematical modeling supported two hypotheses as consistent with these observations: (i) that second-phase virus arises from cells newly infected by long-lived infected cells and (2) that it arises from activation of latently infected cells with full-length unintegrated HIV DNA.These observations challenge the current understanding of HIV-1 turnover and compartmentalization. They also indicate the promise of this new integrase inhibitor raltegravir.

Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4 + T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.