Search

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5-6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape.

Objectives: The evolution of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) necessitates rapid methods for assessing monoclonal antibody (mAb) potency against emerging variants. Authentic virus neutralisation assays are considered the gold standard for measuring virus‐neutralising antibody (nAb) titres in serum. However, authentic virus‐based assays pose inherent practical challenges for measuring nAb titres against emerging SARS‐CoV‐2 variants (e.g. storing infectious viruses and testing at biosafety level‐3 facilities). Here, we demonstrate the utility of pseudovirus neutralisation assay data in conjunction with serum mAb concentrations to robustly predict nAb titres in serum. Methods: SARS‐CoV‐2 nAb titres were determined via authentic‐ and lentiviral pseudovirus‐based neutralisation assays using serological data from three AZD7442 (tixagevimab–cilgavimab) studies: PROVENT (NCT04625725), TACKLE (NCT04723394) and a phase 1 dose‐ranging study (NCT04507256). AZD7442 serum concentrations were assessed using immunocapture. Serum‐based half‐maximal inhibitory concentration (IC50) values were derived from pseudovirus nAb titres and serum mAb concentrations, and compared with in vitro IC50 measurements. Results: nAb titres measured via authentic‐ and lentiviral pseudovirus‐based neutralisation assays were strongly correlated for the ancestral SARS‐CoV‐2 virus and SARS‐CoV‐2 Alpha. Serum AZD7442 concentrations and pseudovirus nAb titres were strongly correlated for multiple SARS‐CoV‐2 variants with all Spearman correlation coefficients ≥ 0.78. Serum‐based IC50 values were similar to in vitro IC50 values for AZD7442, for ancestral SARS‐CoV‐2 and Alpha, Delta, Omicron BA.2 and Omicron BA.4/5 variants. Conclusions: These data highlight that serum mAb concentrations and pseudovirus in vitro IC50 values can be used to rapidly predict nAb titres in serum for emerging and historical SARS‐CoV‐2 variants. [ABSTRACT FROM AUTHOR]

Tenofovir alafenamide (TAF) is a prodrug of the nucleoside reverse transcriptase (RT) inhibitor tenofovir (TFV). Compared to the earlier TFV prodrug, TFV disoproxil fumarate (TDF), TAF achieves more than fourfold‐higher intracellular levels of its active metabolite TFV diphosphate (TFV‐DP) in clinical studies, while achieving a significant reduction of TFV systemic exposure. Resistance to TFV has been well established, with the K65R mutation in RT as the signature mutation. Here we evaluated the in vitro activity of TAF and TDF in patient‐derived HIV‐1 isolates harboring the K65R mutation. Clinical isolates containing K65R were cloned into the pXXLAI construct (n = 42). In vitro phenotypic susceptibility of the constructs to TAF and TDF was evaluated in an MT‐2 cell HIV assay and in viral breakthrough assays modeling physiological concentrations of TAF and TDF. TAF and TDF susceptibility were highly correlated in K65R‐containing mutants, ranging from 2.7‐ to 3.0‐fold (K65R alone) and 1.2‐ to 27.6‐fold (K65R+ other RT mutations) relative to wild‐type. In viral breakthrough assays mimicking differences in physiological concentrations, TAF inhibited breakthrough of 40 of 42 clinical isolates, while the TDF equivalent only inhibited 32 of 42 isolates tested. TAF displayed a higher barrier to resistance than TDF in this panel of K65R‐containing clinical isolates. [ABSTRACT FROM AUTHOR]

Background: Lenacapavir (LEN) is a first-in-class inhibitor of human immunodeficiency virus type 1 (HIV-1) capsid function in clinical development for the treatment of heavily treatment-experienced (HTE) people with HIV (PWH) harboring multidrug resistance (MDR) in combination with an optimized background regimen (OBR). Here we describe resistance analyses conducted in the pivotal phase 2/3 CAPELLA study.Methods: CAPELLA enrolled viremic HTE PWH with resistance to ≥3 of 4 of the main antiretroviral (ARV) classes and resistance to ≥2 ARV drugs per class. Baseline resistance analyses used commercial assays (HIV-1 protease, reverse transcriptase, integrase genotypic/phenotypic tests). Postbaseline resistance was evaluated in participants experiencing virologic failure.Results: At baseline, 46% of participants had resistance to the 4 main ARV drug classes, with one-third of participants having exhausted all drugs from ≥3 of the 4 main ARV classes. Treatment with LEN + OBR for 26 weeks led to viral suppression in 81% of participants. Postbaseline resistance mutations to lenacapavir occurred in 8 participants (6 with M66I, 1 with K70H, 1 with Q67H + K70R) who were receiving unintended functional LEN monotherapy at the time of resistance selection.Conclusions: LEN added to OBR led to high efficacy in this HTE patient population with MDR but could select for resistance when used unintentionally as functional monotherapy. [ABSTRACT FROM AUTHOR]

Introduction: HIV reservoirs and infected cells may persist in tissues with low concentrations of antiretrovirals (ARVs). Traditional pharmacology methods cannot assess variability in ARV concentrations within morphologically complex tissues, such as lymph nodes (LNs). We evaluated the distribution of six ARVs into LNs and the proximity of these ARVs to CD4+ T cells and cell‐associated RT‐SHIV viral RNA. Methods: Between December 2014 and April 2017, RT‐SHIV infected (SHIV+; N = 6) and healthy (SHIV–; N = 6) male rhesus macaques received two selected four‐drug combinations of six ARVs over 10 days to attain steady‐state conditions. Serial cryosections of axillary LN were analysed by a multimodal imaging approach that combined mass spectrometry imaging (MSI) for ARV disposition, RNAscope in situ hybridization for viral RNA (vRNA) and immunohistochemistry for CD4+ T cell and collagen expression. Spatial relationships across these four imaging domains were investigated by nearest neighbour search on co‐registered images using MATLAB. Results: Through MSI, ARV‐dependent, heterogeneous concentrations were observed in different morphological LN regions, such as the follicles and medullary sinuses. After 5–6 weeks of infection, more limited ARV penetration into LN tissue relative to the blood marker heme was found in SHIV+ animals (SHIV+: 0.7 [0.2–1.4] mm; SHIV–: 1.3 [0.5–1.7] mm), suggesting alterations in the microcirculation. However, we found no detectable increase in collagen deposition. Regimen‐wide maps of composite ARV distribution indicated that up to 27% of SHIV+ LN tissue area was not exposed to detectable ARVs. Regions associated with B cell follicles had median 1.15 [0.94–2.69] ‐fold reduction in areas with measurable drug, though differences were only statistically significant for tenofovir (p = 0.03). Median co‐localization of drug with CD4+ target cells and vRNA varied widely by ARV (5.1–100%), but nearest neighbour analysis indicated that up to 10% of target cells and cell‐associated vRNA were not directly contiguous to at least one drug at concentrations greater than the IC50 value. Conclusions: Our investigation of the spatial distributions of drug, virus and target cells underscores the influence of location and microenvironment within LN, where a small population of T cells may remain vulnerable to infection and low‐level viral replication during suppressive ART. [ABSTRACT FROM AUTHOR]

Background: Lenacapavir in vitro resistance selections identified seven mutations in HIV-1 capsid protein (CA) associated with reduced susceptibility.Objectives: To analyse lenacapavir activity against lenacapavir-associated resistance mutations in multiple assays. We also report Day 10 resistance analyses conducted in a Phase 1b study of lenacapavir (Study 4072) in people with HIV (PWH).Methods: Mutations were inserted in a proviral DNA clone by site-directed mutagenesis, and viruses (n = 12) were generated by transfection. Sequences were used to generate single-cycle (SC) test vectors that were evaluated in a Gag-Pro assay, and replicative viruses were tested in a multicycle (MC) MT-2 assay to determine lenacapavir susceptibility. Study 4072 was a Phase 1b, double-blinded, placebo-controlled, dose-ranging, randomized study of lenacapavir in untreated PWH. Participants received a single dose of lenacapavir (up to 750 mg) or placebo (10 day monotherapy). CA resistance was characterized using genotypic and/or phenotypic assays.Results: Lenacapavir susceptibility in the SC assay showed an inverse relationship between replication capacity and resistance. In Study 4072, all 29 participants receiving lenacapavir showed a robust virological response with no rebound. At baseline, no participant had resistance mutations to lenacapavir, and all had WT susceptibility to lenacapavir. Post-monotherapy analyses revealed the emergence of CA mutation Q67H at Day 10 in two participants.Conclusions: In vitro assays confirmed that increased resistance to lenacapavir was associated with decreased replication capacity of mutant viruses. In the clinical study no pre-existing lenacapavir resistance was detected. Emergence of Q67H occurred at exposures below the dose used in current Phase 2/3 studies. These results support development of lenacapavir as an antiretroviral agent. [ABSTRACT FROM AUTHOR]

Background: Routine HIV drug resistance genotyping identified an integrase sequence harbouring T97A, E138K, G140S and Q148H, with high predicted resistance to all integrase strand transfer inhibitors (INSTIs).Objectives: To assess the impact of these substitutions alone and together on phenotypic INSTI susceptibility.Methods: We constructed recombinant NL4.3 viruses harbouring all mutation combinations in the autologous integrase sequence. Viruses were grown in GFP-reporter CD4+ T-cells in the presence of 0.01-1000 nM raltegravir, elvitegravir, dolutegravir, bictegravir, and cabotegravir. Infection was measured by imaging cytometry.Results: Q148H-containing viruses lacking G140S failed to propagate or mutated in vitro, consistent with fitness costs. Statistically significant reductions in INSTI susceptibility were observed for several mutation combinations, as follows. T97A or G140S alone conferred 3.6- to 5.6-fold decreased susceptibility to raltegravir and elvitegravir. Two-mutation combinations conferred low-to-moderate resistance to raltegravir and elvitegravir only, except G140S/Q148H which eliminated raltegravir and elvitegravir activity and conferred 24.6-, 7.9-, and 107.5-fold reduced susceptibility to dolutegravir, bictegravir and cabotegravir. Addition of E138K to G140S/Q148H conferred 35.5, 11.6 and 208-fold reduced susceptibility to dolutegravir, bictegravir, and cabotegravir, while addition of T97A to G140S/Q148H conferred 318, 121 and >1000-fold reduced susceptibility to these drugs. T97A/E138K/G140S/Q148H in the autologous backbone conferred >300-fold reduced susceptibility to all INSTIs. Notably, bictegravir EC50 was significantly lower when T97A/E138K/G140S/Q148H was introduced into NL4.3, suggesting that other mutations in the autologous sequence enhanced resistance.Conclusions: High-level dolutegravir, bictegravir and cabotegravir resistance requires multiple integrase substitutions including compensatory mutations. T97A and E138K further enhance the resistance conferred by G140S/Q148H, yielding >300-fold decreased susceptibility to all INSTIs when all four mutations are present. [ABSTRACT FROM AUTHOR]

In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants. Author summary: The global employment of mRNA-based COVID-19 vaccines, together with their high protection, has enabled to control the pandemic in places with high vaccination rate. However, the recent increase in the frequency of SARS-CoV-2 variants with reduced sensitivity to antibody neutralization has raised concerns about the ability of COVID-19 vaccines to eradicate SARS-CoV-2. Despite in-depth characterization of such variants, little is known about SARS-CoV-2 evolution within the host. Here, we preformed in-depth analysis of swab samples isolated from infected individuals and demonstrated that intra-host variants with reduced infectivity are developed during the course of the infection. Furthermore, these variants have varied sensitivity to the BNT162b2 vaccine and to convalescent plasma. In addition, we identified a point mutation at the S2 subunit of the spike protein, which impairs antibody neutralization activity. Our findings shed light on SARS-CoV-2 adaptation to the human host and highlight the importance of mapping the landscape of SARS-CoV-2 intra-host variability for better evaluating variants of concern. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection. [ABSTRACT FROM AUTHOR]

Background: Fostemsavir is a prodrug of a first-in-class HIV-1 attachment inhibitor, temsavir, that binds to gp120 and blocks attachment to the host-cell CD4 receptor, preventing entry and infection of the target cell. Previous studies using a limited number of clinical isolates showed that there was intrinsic variability in their susceptibility to temsavir.Objectives: Here, an analysis was performed using all clinical isolates analysed in the Monogram Biosciences PhenoSense® Entry assay as part of the development programme.Methods: In total, 1337 individual envelopes encompassing 20 different HIV-1 subtypes were examined for their susceptibility to temsavir. However, only seven subtypes (B, C, F1, A, [B, F1], BF and A1) were present more than five times, with subtype B (881 isolates) and subtype C (156 isolates) having the largest numbers.Results: As expected, variability in susceptibility was observed within all subtypes. However, for the great majority of these viruses, temsavir was highly potent, with most viruses exhibiting IC50s <10 nM. One exception was CRF01_AE viruses, where all five isolates exhibited IC50s >100 nM. For the 607 isolates where tropism data were available, geometric mean temsavir IC50 values were remarkably similar for CCR5-, CXCR4- and dual mixed-tropic envelopes from infected individuals.Conclusions: These data show that HIV-1 viruses from most subtypes are highly susceptible to temsavir and that temsavir susceptibility is independent of tropism. [ABSTRACT FROM AUTHOR]

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies. [ABSTRACT FROM AUTHOR]

Background: HIV-1 can develop resistance to antiretroviral drugs, mainly through mutations within the target regions of the drugs. In HIV-1 protease, a majority of resistance-associated mutations that develop in response to therapy with protease inhibitors are found in the protease's active site that serves also as a binding pocket for the protease inhibitors, thus directly impacting the protease-inhibitor interactions. Some resistance-associated mutations, however, are found in more distant regions, and the exact mechanisms how these mutations affect protease-inhibitor interactions are unclear. Furthermore, some of these mutations, e.g. N88S and L76V, do not only induce resistance to the currently administered drugs, but contrarily induce sensitivity towards other drugs. In this study, mutations N88S and L76V, along with three other resistance-associated mutations, M46I, I50L, and I84V, are analysed by means of molecular dynamics simulations to investigate their role in complexes of the protease with different inhibitors and in different background sequence contexts. Results: Using these simulations for alchemical calculations to estimate the effects of mutations M46I, I50L, I84V, N88S, and L76V on binding free energies shows they are in general in line with the mutations' effect on I C 50 values. For the primary mutation L76V, however, the presence of a background mutation M46I in our analysis influences whether the unfavourable effect of L76V on inhibitor binding is sufficient to outweigh the accompanying reduction in catalytic activity of the protease. Finally, we show that L76V and N88S changes the hydrogen bond stability of these residues with residues D30/K45 and D30/T31/T74, respectively. Conclusions: We demonstrate that estimating the effect of both binding pocket and distant mutations on inhibitor binding free energy using alchemical calculations can reproduce their effect on the experimentally measured I C 50 values. We show that distant site mutations L76V and N88S affect the hydrogen bond network in the protease's active site, which offers an explanation for the indirect effect of these mutations on inhibitor binding. This work thus provides valuable insights on interplay between primary and background mutations and mechanisms how they affect inhibitor binding. [ABSTRACT FROM AUTHOR]

The HIV‐1 protease is a major target of inhibitor drugs in AIDS therapies. The therapies are impaired by mutations of the HIV‐1 protease that can lead to resistance to protease inhibitors. These mutations are classified into major mutations, which usually occur first and clearly reduce the susceptibility to protease inhibitors, and minor, accessory mutations that occur later and individually do not substantially affect the susceptibility to inhibitors. Major mutations are predominantly located in the active site of the HIV‐1 protease and can directly interfere with inhibitor binding. Minor mutations, in contrast, are typically located distal to the active site. A central question is how these distal mutations contribute to resistance development. In this article, we present a systematic computational investigation of stability changes caused by major and minor mutations of the HIV‐1 protease. As most small single‐domain proteins, the HIV‐1 protease is only marginally stable. Mutations that destabilize the folded, active state of the protease therefore can shift the conformational equilibrium towards the unfolded, inactive state. We find that the most frequent major mutations destabilize the HIV‐1 protease, whereas roughly half of the frequent minor mutations are stabilizing. An analysis of protease sequences from patients in treatment indicates that the stabilizing minor mutations are frequently correlated with destabilizing major mutations, and that highly resistant HIV‐1 proteases exhibit significant fractions of stabilizing mutations. Our results thus indicate a central role of minor mutations in balancing the marginal stability of the protease against the destabilization induced by the most frequent major mutations. [ABSTRACT FROM AUTHOR]

Background: The rate of S68G mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has increased and is closely related to the K65R mutation among CRF01_AE-infected patients who failed treatment. We aimed to explore the temporal association of S68G and K65R mutations and disclose the role of the former on susceptibility to nucleotide/nucleoside reverse transcriptase inhibitor (NRTI) and viral replication with the K65R double mutations among CRF01_AE-infected patients who failed treatment.Methods: The occurrence of S68G and K65R mutations was evaluated among HIV-1 of various subtypes in the global HIV Drug Resistance Database. The temporal association of S68G and K65R mutations was analyzed through next-generation sequencing in four CRF01_AE-infected patients who failed treatment with tenofovir/lamivudine/efavirenz. The impact of the S68G mutation on susceptibility to NRTI and replication fitness was analyzed using pseudovirus phenotypic resistance assays and growth competition assays, respectively.Results: The frequency of the S68G mutation increased by 1.4-9.7% in almost all HIV subtypes and circulating recombinant forms in treatment-experienced patients, except subtype F. The S68G mutation often occurred in conjunction with the K65R mutation among RTI-treated patients, with frequencies ranging 21.1-61.7% in various subtypes. Next-generation sequencing revealed that the S68G mutation occurred following the K65R mutation in three of the four CRF01_AE-infected patients. In these three patients, there was no significant change detected in the half maximal inhibitory concentration for zidovudine, tenofovir, or lamivudine between the K65R and K65R/S68G mutations, as demonstrated by the phenotypic resistance assays. Virus stocks of the K65R and K65R/S68G mutations were mixed with 4:6, 1:1, and 9:1 and cultured for 13 days, the K65R/S68G mutants outgrew those of the K65R mutants irrespective of the input ratio.Conclusions: S68G may be a natural polymorphism and compensatory mutation of K65R selected by NRTIs in the CRF01_AE strain of HIV-1. This mutation does not affect susceptibility to NRTI; however, it improves the replication fitness of K65R mutants. This study deciphers the role of the S68G mutation in the HIV reverse transcriptase of the CRF01_AE strain and provides new evidence for the interpretation of drug-resistant mutations in non-B subtypes of HIV-1. [ABSTRACT FROM AUTHOR]

GSK3532795 (formerly BMS955176) is a second-generation maturation inhibitor (MI) that progressed through a Phase 2b study for treatment of HIV-1 infection. Resistance development to GSK3532795 was evaluated through in vitro methods and was correlated with information obtained in a Phase 2a proof-of-concept study in HIV-1 infected participants. Both low and high concentrations of GSK3532795 were used for selections in vitro, and reduced susceptibility to GSK3532795 mapped specifically to amino acids near the capsid/ spacer peptide 1 (SP1) junction, the cleavage of which is blocked by MIs. Two key substitutions, A364V or V362I, were selected, the latter requiring secondary substitutions to reduce susceptibility to GSK3532795. Three main types of secondary substitutions were observed, none of which reduced GSK3532795 susceptibility in isolation. The first type was in the capsid C-terminal domain and downstream SP1 region (including (Gag numbering) R286K, A326T, T332S/N, I333V and V370A/M). The second, was an R41G substitution in viral protease that occurred with V362I. The third was seen in the capsid N-terminal domain, within the cyclophilin A binding domain (V218A/M, H219Q and G221E). H219Q increased viral replication capacity and reduced susceptibility of poorly growing viruses. In the Phase 2a study, a subset of these substitutions was also observed at baseline and some were selected following GSK35323795 treatment in HIV-1-infected participants. [ABSTRACT FROM AUTHOR]

1. Antiretroviral concentrations in cerebrospinal fluid (CSF) are used as surrogate for brain tissue, although sparse data support this. We quantified antiretrovirals in brain tissue across preclinical models, compared them to CSF, and calculated 90% inhibitory quotients (IQ90) for nonhuman primate (NHP) brain tissue. Spatial distribution of efavirenz was performed by mass-spectrometry imaging (MSI). 2. HIV or RT-SHIV-infected and uninfected animals from two humanized mouse models (hemopoietic-stem cell/RAG2-, n = 36; bone marrow-liver-thymus/BLT, n =13) and an NHP model (rhesus macaque, n =18) were dosed with six antiretrovirals. Brain tissue, CSF (NHPs), and plasma were collected at necropsy. Drug concentrations were measured by LC-MS/MS. Rapid equilibrium dialysis determined protein binding in NHP brain. 3. Brain tissue penetration of most antiretrovirals were >10-fold lower (p < 0.02) in humanized mice than NHPs. NHP CSF concentrations were >13-fold lower (p <0.02) than brain tissue with poor agreement except for efavirenz (r = 0.91, p = 0.001). Despite 97% brain tissue protein binding, efavirenz achieved IQ90>1 in all animals and 2-fold greater white versus gray matter concentration. 4. Brain tissue penetration varied across animal models for all antiretrovirals except raltegravir, and extrapolating brain tissue concentrations between models should be avoided. With the exception of efavirenz, CSF is not a surrogate for brain tissue concentrations. [ABSTRACT FROM AUTHOR]

Background: Dual therapy combining integrase inhibitors and NNRTIs represents a promising regimen in ageing HIV-infected individuals with long exposure to nucleoside analogues and PIs.Methods: The ANRS 163 ETRAL trial (NCT02212379) was a 96 week, multicentre, single-arm study evaluating the efficacy and safety of raltegravir (400 mg twice daily)/etravirine (200 mg twice daily) in individuals >45 years, on a PI-containing regimen who were integrase inhibitor and etravirine naive. The primary endpoint was the proportion of participants with virological success, defined by the absence of virological failure up to week 48. Main secondary outcomes included evolution of metabolic parameters, CD4/CD8 count, bone mineral density and inflammatory markers. The study was designed to show an efficacy >90%, assuming a success rate ≥95%, with a power of 80% and a 5% type-1 error.Results: One hundred and sixty-five participants (median age 52 years, duration of ART 16.9 years, viral suppression 6.9 years and CD4 count 700 cells/mm3) were enrolled. By ITT analysis, viral suppression was maintained in 99.4% of participants (95% CI = 95.6%-99.9%) at week 48 and 98.7% (95% CI = 95.0%-99.7%) at week 96. Two virological failures occurred (week 24 and week 64) without emergence of integrase inhibitor resistance. Eight participants discontinued raltegravir/etravirine for adverse events, leading to a strategy success rate of 95.1% (95% CI = 90.5%-97.5%) at week 48 and 92.7% (95% CI = 87.5%-95.8%) at week 96. Over 96 weeks, lipid fractions improved (P < 0.001), CD4/CD8 ratio increased, IFNγ-induced protein 10 (IP-10) decreased (-8.1%), soluble CD14 decreased (-27%, P < 0.001) bone mineral density improved and BMI increased.Conclusions: Raltegravir plus etravirine dual therapy demonstrated durable efficacy in virologically suppressed ageing patients. [ABSTRACT FROM AUTHOR]

Study Objective: The ANRS163‐ETRAL study showed that etravirine 200 mg/raltegravir 400 mg twice‐daily dual therapy was highly effective in the treatment of human immunodeficiency virus (HIV)‐infected patients older than 45 years, with virologic and therapeutic success rates at week 48 of 99.4% and 94.5%, respectively. The objective of this study was to determine whether a clinically significant pharmacokinetic interaction between etravirine and raltegravir exists by assessing steady‐state total and unbound etravirine, raltegravir, and inactive raltegravir‐glucuronide concentrations 12 hours after last intake (C12h) in blood plasma (BP) and seminal plasma (SP). Design: Pharmacokinetic analysis of data from the ANRS163‐ETRAL study. Patients: One hundred forty‐six HIV‐1–infected patients (of the 165 patients included in the ANRS‐163 ETRAL study) who were receiving etravirine 200 mg and raltegravir 400 mg twice daily. Measurements and Main Results: Blood was collected from all 146 patients at weeks 2–4, 12, 24, and 48, and semen was collected from 21 patients at week 48. The extent of BP and SP protein binding was determined by using ultrafiltration assay. Total and unbound etravirine, raltegravir, and raltegravir‐glucuronide C12h were determined by ultra high performance liquid chromatography coupled with tandem mass spectrometry and interpreted by using the in vitro calculated protein‐bound 95% inhibitory concentration (PBIC95) for wild‐type (WT) HIV: etravirine (116 ng/ml) and raltegravir (15 ng/ml). Median (interquartile range [IQR]) total BP etravirine C12h (536 ng/ml [376–719]) and raltegravir (278 ng/ml [97–690]) were adequate in 99% and 96% of patients, respectively. Median (IQR) SP:BP C12h ratio and BP unbound fraction were etravirine 0.3 (0.2–0.5) and < 1%, respectively, raltegravir 1.8 (1.3–3.3) and 12%, respectively, and raltegravir‐glucuronide 12.0 (6.5–17.7) and > 99%, respectively. The BP raltegravir metabolic ratio (raltegravir glucuronide:raltegravir ratio) was 1.7, suggesting only weak induction of raltegravir glucuronidation by etravirine. Only three patients had etravirine and raltegravir C12h < PBIC95 simultaneously. Conclusion: No clinically significant pharmacokinetic interaction between etravirine and raltegravir was detected. Total etravirine and raltegravir BP concentrations were adequate in most patients, favoring virologic efficacy and confirming good treatment adherence (> 95%), despite twice‐daily administration. The long half‐life of etravirine and higher unbound fraction SP of raltegravir (57%) ensured adequate concentrations of dual therapy in genital compartments. Our results indicate that etravirine and raltegravir have good, complementary pharmacokinetic profiles, suggesting that they could be used in a dual‐treatment strategy. [ABSTRACT FROM AUTHOR]

Objectives: The WHO recently recommended the use of a new first-line ART containing dolutegravir. We investigated the efficacy of NRTI backbones (tenofovir or abacavir with a cytosine analogue) in low- and middle-income countries where there is significant prior exposure to antiretrovirals and drug resistance to NRTIs.Methods: Within the treatment-as-prevention study in South Africa, we selected participants with available next-generation sequencing (NGS) data for the HIV-1 pol gene at trial entry; they were either ART initiators (n = 1193) or already established on ART (n = 94). NGS of the HIV-1 pol gene was carried out using MiSeq technology; reverse transcriptase drug resistance mutations (DRMs) were detected at 5% (DRM5%) and 20% (DRM20%) for all 1287 participants. Genotypic susceptibility was assessed using the Stanford HIVDB resistance interpretation algorithm.Results: NRTI DRM20% and DRM5% were detected among 5/1193 (0.4%) and 9/1193 (0.8%) of ART initiators, respectively. There was tenofovir exposure in 73/94 (77.7%) of those established on ART, with full susceptibility to abacavir in 57/94 (60.6%) and 56/94 (59.6%) for DRM20% and DRM5%, respectively, while 67/94 (71.3%) and 64/94 (68.1%) were fully susceptible to tenofovir, respectively. The differences between tenofovir and abacavir were not statistically significant at the 20% or 5% variant level (P = 0.16 and 0.29, respectively). NGS detection of variants at the 5% level increased detection of K65R in both naive and treated groups. One of 607 integrase sequences carried a DRM20% (Q148R).Conclusions: Dolutegravir with a cytosine analogue plus tenofovir or abacavir appears to have similar efficacy in South Africans naive to ART. NGS should be considered in HIV drug resistance surveillance. [ABSTRACT FROM AUTHOR]

Background Contemporary antiretroviral therapies (ART) and management strategies have diminished both human immunodeficiency virus (HIV) treatment failure and the acquired resistance to drugs in resource-rich regions, but transmission of drug-resistant viruses has not similarly decreased. In low- and middle-income regions, ART roll-out has improved outcomes, but has resulted in increasing acquired and transmitted resistances. Our objective was to review resistance to ART drugs and methods to detect it, and to provide updated recommendations for testing and monitoring for drug resistance in HIV-infected individuals. Methods A volunteer panel of experts appointed by the International Antiviral (formerly AIDS) Society–USA reviewed relevant peer-reviewed data that were published or presented at scientific conferences. Recommendations were rated according to the strength of the recommendation and quality of the evidence, and reached by full panel consensus. Results Resistance testing remains a cornerstone of ART. It is recommended in newly-diagnosed individuals and in patients in whom ART has failed. Testing for transmitted integrase strand-transfer inhibitor resistance is currently not recommended, but this may change as more resistance emerges with widespread use. Sanger-based and next-generation sequencing approaches are each suited for genotypic testing. Testing for minority variants harboring drug resistance may only be considered if treatments depend on a first-generation nonnucleoside analogue reverse transcriptase inhibitor. Different HIV-1 subtypes do not need special considerations regarding resistance testing. Conclusions Testing for HIV drug resistance in drug-naive individuals and in patients in whom antiretroviral drugs are failing, and the appreciation of the role of testing, are crucial to the prevention and management of failure of ART. [ABSTRACT FROM AUTHOR]

The biology of HIV is rather complex due to high rate of replication, frequent recombination, and introduction of mutations. This gives rise to a number of distinct variants referred as quasispecies. In addition, the latency within reservoir allows the periodic reactivation of virus replication. The rapid replication of HIV allows immune response escape and establishment of resistance to therapy that can be acquired through drug selection and/or transmitted among individuals. This prompted, over the years, the development of a range of assays aimed to determine drug resistance and sensitivity, to be used both in clinical practice and in antiviral research. Reverse transcriptase (RT) inhibitors have an eminent place among the anti-HIV drugs, being constantly present from the beginning until today in the most commonly used antiviral regimens. This mini-review seeks to provide an up-to-date overview of recent efforts in developing even more reliable and simple methods, of both genotypic and phenotypic types, for specifically detecting drug resistance and sensitivity to RT inhibitors. [ABSTRACT FROM AUTHOR]

Background: Reducing the dose of efavirenz can improve safety, reduce costs, and increase access for patients with HIV infection. According to the World Health Organization, a similar dosing strategy for all patient populations is desirable for universal roll-out; however, it remains unknown whether the 400 mg daily dose is adequate during pregnancy.Methods: We developed a mechanistic population pharmacokinetic model using pooled data from women included in seven studies (1968 samples, 774 collected during pregnancy). Total and free efavirenz exposure (AUC24 and C12) were predicted for 400 (reduced) and 600 mg (standard) doses in both pregnant and non-pregnant women.Results: Using a 400 mg dose, the median efavirenz total AUC24 and C12 during the third trimester of pregnancy were 91 and 87% of values among non-pregnant women, respectively. Furthermore, the median free efavirenz C12 and AUC24 were predicted to increase during pregnancy by 11 and 15%, respectively.Conclusions: It was predicted that reduced-dose efavirenz provides adequate exposure during pregnancy. These findings warrant prospective confirmation. [ABSTRACT FROM AUTHOR]

Objectives: 4'-Ethnyl-2'-fluoro-2'-deoxyadenosine (EFdA) is a novel translocation-defective reverse transcriptase inhibitor. We investigated the virological and biochemical inhibitory potentials of EFdA against a broad spectrum of subtype-specific chimeric viruses and compared it with tenofovir alafenamide, nevirapine, efavirenz, rilpivirine and etravirine.Methods: pNL4.3 chimeric viruses encoding gag-pol from treatment-naive patients (n = 24) and therapy-failure patients (n = 3) and a panel of reverse transcriptase inhibitor-resistant strains (n = 7) were used to compare the potency of reverse transcriptase inhibitor drugs. The phenotypic drug susceptibility assay was performed using TZM-bl cells. In vitro inhibition assays were done using patient-derived reverse transcriptase. IC50 values of NNRTIs were calculated using a PicoGreen-based spectrophotometric assay. Steady-state kinetics were used to determine the apparent binding affinity (Km.dNTP) of triphosphate form of EFdA (EFdA-TP) and dATP.Results: Among the chimeric treatment-naive viruses, EFdA had an ex vivo antiretroviral activity [median (IQR) EC50 = 1.4 nM (0.6-2.1 nM)] comparable to that of tenofovir alafenamide [1.6 nM (0.5-3.6 nM)]. Subtype-specific differences were found for etravirine (P = 0.004) and rilpivirine (P = 0.017), where HIV-1C had the highest EC50 values. EFdA had a greater comparative efficiency [calculated by dividing the efficiency of monophosphate form of EFdA (EFdA-MP) incorporation (kcat.EFdA-TP/Km.EFdA-TP) over the efficiency of dATP incorporation (kcat.dATP/Km.dATP)] compared with the natural substrate dATP, with a fold change of between 1.6 and 3.2. Ex vivo analysis on reverse transcriptase inhibitor-resistant strains showed EFdA to have a higher potency. Despite the presence of rilpivirine DRMs, some non-B strains showed hypersusceptibility to rilpivirine.Conclusions: Our combined virological and biochemical data suggest that EFdA inhibits both WT and reverse transcriptase inhibitor-resistant viruses efficiently in a subtype-independent manner. In contrast, HIV-1C is least susceptible to etravirine and rilpivirine. [ABSTRACT FROM AUTHOR]

Most HIV-infected patients who initiate combination antiretroviral therapy experience a viral load decline in several phases. These phases are characterized by different rates of viral load decay that decrease when transitioning from one phase to the next. There is no consensus as to the origin of these phases. One hypothesis put forward is that short- and long-lived infected cells are responsible for the first and second phases of decay, respectively. However, significant differences in drug concentrations are observed in monocytes from various tissues, suggesting the first two phases of decay in viral loads could instead be attributed to short-lived cells being differently exposed to drugs. Compared to a well-exposed compartment, new cell infection can be expected in a compartment with limited drug exposure, thus leading to a slower viral load decay with potential virologic failure and drug resistance. In the current study, the latter hypothesis was investigated using a model of viral kinetics. Empirical datasets were involved in model elaboration and parameter estimation. In particular, susceptibility assay data was used for an in vitro to in vivo extrapolation based on the expected drug concentrations inside physiological compartments. Results from numerical experiments of the short-term evolution of viral loads can reproduce the first two phases of viral decay when allowing new short-lived cell infections in an unidentified drug-limited compartment. Model long-term predictions are however less consistent with clinical observations. For the hypothesis to hold, efavirenz, tenofovir and emtricitabine drug exposure in the drug-limited compartment would have to be very low compared to exposure in peripheral blood. This would lead to significant long-term viral growth and the frequent development of resistant strains, a prediction not supported by clinical observations. This suggests that the existence of a drug-limited anatomical compartment is unlikely, by itself, to explain the second phase of viral load decay. [ABSTRACT FROM AUTHOR]

The evolution of viral pathogens is shaped by strong selective forces that are exerted during jumps to new hosts, confrontations with host immune responses and antiviral drugs, and numerous other processes. However, while undeniably strong and frequent, adaptive evolution is largely confined to small parts of information-packed viral genomes, and the majority of observed variation is effectively neutral. The predictions and implications of the neutral theory have proven immensely useful in this context, with applications spanning understanding within-host population structure, tracing the origins and spread of viral pathogens, predicting evolutionary dynamics, andmodeling the emergence of drug resistance. We highlight the multiple ways in which the neutral theory has had an impact, which has been accelerated in the age of high-throughput, high-resolution genomics. [ABSTRACT FROM AUTHOR]

Background: Paediatric data on CNS penetration of antiretroviral drugs are scarce.Objectives: To evaluate CNS penetration of antiretroviral drugs in HIV-infected children and explore associations with neurocognitive function.Patients and methods: Antiretroviral drug levels were measured in paired CSF and blood samples of clinically stable HIV-infected children between 8 and 18 years old on long-term combined ART. Plasma drug concentrations were corrected for protein binding. We evaluated CNS penetration using CSF/plasma ratios and compared CSF concentrations with the IC50 as a surrogate marker for effectiveness. Blood-brain barrier permeability was assessed for possible confounding. Associations with neurocognitive function were explored using linear regression analysis.Results: Median CSF/plasma ratios (IQR) were: lopinavir 0.059 (0.024-0.157, n = 7), efavirenz 0.681 (0.555-0.819, n = 12), tenofovir 0.021 (0.020-0.024, n = 4), lamivudine 0.464 (0.331-0.607, n = 17), emtricitabine 0.365 (0.343-0.435, n = 3), nevirapine 1.203 (n = 1), zidovudine 0.718 (0.711-1.227, n = 5) and abacavir 1.344 (0.670-2.450, n = 10). CSF concentrations were below the IC50 for tenofovir (100%), emtricitabine (100%), abacavir (50%) and zidovudine (17%). Lamivudine, lopinavir, efavirenz and nevirapine concentrations were all above the IC50. All participants were virologically suppressed in blood and CSF. CSF drug concentrations were not associated with blood-brain barrier permeability or neurocognitive function.Conclusions: We showed adequate CSF concentrations of lamivudine, lopinavir, efavirenz and nevirapine, and potential suboptimal CSF concentrations of tenofovir, abacavir and emtricitabine in long-term treated HIV-infected children. None the less, the use of combined antiretroviral drugs led to adequate viral suppression. [ABSTRACT FROM AUTHOR]

Background and Objectives: Long-acting injectable antiretrovirals represent a pharmacological alternative to oral formulations and an innovative clinical option to address adherence and reduce drug costs. Clinical studies in children and adolescents are characterised by ethical and logistic barriers complicating the identification of dose optimisation. Physiologically-based pharmacokinetic modelling represents a valuable tool to inform dose finding prior to clinical trials. The objective of this study was to simulate potential dosing strategies for existing long-acting injectable depot formulations of cabotegravir and rilpivirine in children and adolescents (aged 3-18 years) using physiologically-based pharmacokinetic modelling.Methods: Whole-body physiologically-based pharmacokinetic models were developed to represent the anatomical, physiological and molecular processes and age-related changes in children and adolescents through allometric equations. Models were validated for long-acting injectable intramuscular cabotegravir and rilpivirine in adults. Subsequently, the anatomy and physiology of children and adolescents were validated against available literature. The optimal doses of monthly administration of cabotegravir and rilpivirine were identified in children and adolescents, to achieve trough concentrations over the target concentrations derived in a recent efficacy trial of the same formulations.Results: Pharmacokinetic data generated through the physiologically-based pharmacokinetic simulations were similar to observed clinical data in adults. Optimal doses of long-acting injectable antiretrovirals cabotegravir and rilpivirine were predicted using the release rate observed for existing clinical formulations, for different weight groups of children and adolescents. The intramuscular loading dose and maintenance dose of cabotegravir ranged from 200 to 600 mg and from 100 to 250 mg, respectively, and for rilpivirine it ranged from 250 to 550 mg and from 150 to 500 mg, respectively, across various weight groups of children ranging from 15 to 70 kg.Conclusions: The reported findings represent a rational platform for the identification of suitable dosing strategies and can inform prospective clinical investigation of long-acting injectable formulations in children and adolescents. [ABSTRACT FROM AUTHOR]

Background: MK-8507 is a novel HIV-1 non-nucleoside reverse transcriptase inhibitor being developed for treatment of HIV-1 infection. MK-8507 has high antiviral potency in vitro and pharmacokinetic (PK) properties that support once-weekly dosing., Setting: A phase 1, open-label, proof-of-concept study was conducted in treatment-naive adults with HIV-1 infection to assess monotherapy antiviral activity., Methods: In 3 sequential panels, participants aged 18-60 years with baseline plasma HIV-1 RNA ≥10,000 copies/mL and CD4+ T-cell count >200/mm3 received a single oral dose of 40, 80, or 600 mg MK-8507 in the fasted state. Participants were assessed for HIV-1 RNA for at least 7 days, PKs for 14 days, and safety and tolerability for 21 days postdose., Results: A total of 18 participants were enrolled (6 per panel). The mean 7-day postdose HIV-1 RNA reduction ranged from ∼1.2 to ∼1.5 log10 copies/mL across the doses assessed. One patient had a viral rebound associated with emergence of an F227C reverse transcriptase variant (per chain-termination method sequencing) 14 days postdose; this variant was found in a second participant by ultra-deep sequencing as an emerging minority variant. MK-8507 PKs were generally dose-proportional and similar to observations in participants without HIV-1 infection in prior studies; mean MK-8507 half life was 56-69 hours in this study. MK-8507 was generally well tolerated at all doses., Conclusions: The robust antiviral activity, PK, and tolerability of MK-8507 support its continued development as part of a complete once weekly oral regimen for HIV-1 treatment; combination therapy could mitigate the emergence of resistance-associated variants., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction HIV-1 genotypic resistance test (GRT) interpretation systems (IS) require updates as new studies on HIV-1 drug resistance are published and as treatment guidelines evolve. Methods An expert panel was created to provide recommendations for the update of the Stanford HIV Drug Resistance Database (HIVDB) GRT-IS. The panel was polled on the ARVs to be included in a GRT report, and the drug-resistance interpretations associated with 160 drug-resistance mutation (DRM) pattern-ARV combinations. The DRM pattern-ARV combinations included 52 nucleoside RT inhibitor (NRTI) DRM pattern-ARV combinations (13 patterns x 4 NRTIs), 27 nonnucleoside RT inhibitor (NNRTI) DRM pattern-ARV combinations (9 patterns x 3 NNRTIs), 39 protease inhibitor (PI) DRM pattern-ARV combinations (13 patterns x 3 PIs) and 42 integrase strand transfer inhibitor (INSTI) DRM pattern-ARV combinations (14 patterns x 3 INSTIs). Results There was universal agreement that a GRT report should include the NRTIs lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir disoproxil fumarate; the NNRTIs efavirenz, etravirine, nevirapine, and rilpivirine; the PIs atazanavir/r, darunavir/r, and lopinavir/ r (with "/r" indicating pharmacological boosting with ritonavir or cobicistat); and the INSTIs dolutegravir, elvitegravir, and raltegravir. There was a range of opinion as to whether the NRTIs stavudine and didanosine and the PIs nelfinavir, indinavir/r, saquinavir/r, fosamprenavir/r, and tipranavir/r should be included. The expert panel members provided highly concordant DRM pattern-ARV interpretations with only 6% of NRTI, 6% of NNRTI, 5% of PI, and 3% of INSTI individual expert interpretations differing from the expert panel median by more than one resistance level. The expert panel median differed from the HIVDB 7.0 GRTIS for 20 (12.5%) of the 160 DRM pattern-ARV combinations including 12 NRTI, two NNRTI, and six INSTI pattern-ARV combinations. Eighteen of these differences were updated in HIVDB 8.1 GRT-IS to reflect the expert panel median. Additionally, HIVDB users are now provided with the option to exclude those ARVs not considered to be universally required. Conclusions The HIVDB GRT-IS was updated through a collaborative process to reflect changes in HIV drug resistance knowledge, treatment guidelines, and expert opinion. Such a process broadens consensus among experts and identifies areas requiring further study. [ABSTRACT FROM AUTHOR]

In recent years, the patterns of human immunodeficiency virus 1 (HIV-1) transmission in Iran have been changing gradually from drug injection to unprotected sexual contact. This study sought to investigate the phylogenetic trends and characteristics of transmitted drug resistance (TDR) mutations of HIV-1 in a population that is mainly infected through homo/heterosexual contacts. Sixty newly diagnosed antiretroviral-naive individuals with HIV infection living in Tehran were recruited to this survey, and among them, 42 subjects were established to be infected through sexual intercourse. Following amplification and sequencing of the main part of the HIV-1 pol region, phylogenetic and drug-resistance mutation (DRM) analysis was successfully performed on these 42 patients. Phylogenetic analysis showed that the majority of the subjects were infected with subtype CRF35_AD (88%), followed by subtype B, with 7.1%, and subtype CRF01_AE, with 4.7%. A total of 7.1% of the subjects were found to be infected with HIV-1 variants with surveillance drug-resistant mutations (SDRMs) according to the last world health organisation (WHO) algorithm. All of the identified SDRMs belonged to the non-nucleoside reverse transcriptase inhibitors (NNRTIs) class, including K103 N and V106A, which were found in three patients. Two minor HIV protease-inhibitor-related mutations (L10I and G73S) were detected in two patients, but these mutations are not included in the WHO SDRMs list. The dominance of HIV-1 subtype CRF35_AD was observed among subjects of this study who were infected through sexual contact. The moderate prevalence of SDRMs (7.1%) in this population emphasises the fact that the risk of treatment failure in HIV-infected individuals might increase in the future, and preventive measures should be considered by health authorities. [ABSTRACT FROM AUTHOR]

Antiretroviral treatment history and past HIV-1 genotypes have been shown to be useful predictors for the success of antiretroviral therapy. However, this information may be unavailable or inaccurate, particularly for patients with multiple treatment lines often attending different clinics. We trained statistical models for predicting drug exposure from current HIV-1 genotype. These models were trained on 63,742 HIV-1 nucleotide sequences derived from patients with known therapeutic history, and on 6,836 genotype-phenotype pairs (GPPs). The mean performance regarding prediction of drug exposure on two test sets was 0.78 and 0.76 (ROC-AUC), respectively. The mean correlation to phenotypic resistance in GPPs was 0.51 (PhenoSense) and 0.46 (Antivirogram). Performance on prediction of therapy-success on two test sets based on genetic susceptibility scores was 0.71 and 0.63 (ROC-AUC), respectively. Compared to geno2pheno[resistance], our novel models display a similar or superior performance. Our models are freely available on the internet via . They can be used for inferring which drug compounds have previously been used by an HIV-1-infected patient, for predicting drug resistance, and for selecting an optimal antiretroviral therapy. Our data-driven models can be periodically retrained without expert intervention as clinical HIV-1 databases are updated and therefore reduce our dependency on hard-to-obtain GPPs. [ABSTRACT FROM AUTHOR]

Background.: The presence of transmitted drug-resistance mutations (TDRM) in antiretroviral (ARV) treatment-naïve patients can adversely affect the outcome of ARV therapy.Methods.: Resistance testing was conducted in 6704 ARV-naïve subjects predominantly from the U.S. and Europe in 9 Gilead clinical studies from 2000 to 2013.Results.: The presence of TDRM increased during this period (5.2% to 11.4%), primarily driven by non-nucleoside RT inhibitor resistance mutations (NNRTI; 0.3% to 7.1%), particularly K103N/S (0.3% to 5.3%). NRTI mutations were found in 3.1% of patients. Only 1 patient had K65R (0.01%) and 7 patients had M184V/I (0.1%) despite high use of tenofovir disoproxil fumarate (TDF), emtricitabine, and lamivudine and potential transmission of resistance to these drugs. One or more thymidine analog mutations (TAMs) were present in 2.7% of patients with 0.07% harboring T215Y/F and 2.7% harboring T215 revertant mutations (T215rev). Patients with the combination of M41L+L210W+T215rev showed full HIV RNA suppression while receiving a TDF- or tenofovir alafenamide (TAF)-containing regimen.Conclusions.: There was an overall increase of TDRMs among patients enrolling in clinical trials from 2000 through 2013 driven primarily by an increase in NNRTI-R. However, the presence of common TDRMs, including TAMs/T215rev, showed no impact on response to TDF- or TAF-containing regimens. [ABSTRACT FROM AUTHOR]

Background: HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established., Methods: Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database., Results: Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies., Conclusions: The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs., Competing Interests: B.M., A.P., R.M., C.P., M.B., N.T., S.C., R.G., and C.C. are employees and stockholders of Gilead Sciences. H.F.G. has received unrestricted research grants from Gilead Sciences and Roche; fees for data and safety monitoring board membership from Merck; consulting/advisory board membership fees from Gilead Sciences, Merck, and ViiV Healthcare; and grants from SystemsX and the National Institutes of Health. The institution of H.F.G. received educational grants from Gilead Sciences, ViiV, MSD, Abbvie, and Sandoz. D.L.B. received honoraria and travel grants from Gilead, ViiV, and Merck outside of the submitted work. The remaining authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The genotype-phenotype (GP) map is a central concept in evolutionary biology as it describes the mapping of molecular genetic variation onto phenotypic trait variation. Our understanding of that mapping remains partial, especially when trying to link functional clustering of pleiotropic gene effects with patterns of phenotypic trait co-variation. Only on rare occasions have studies been able to fully explore that link and tend to show poor correspondence between modular structures within the GP map and among phenotypes. By dissecting the structure of the GP map of the replicative capacity of HIV-1 in 15 drug environments, we provide a detailed view of that mapping from mutational pleiotropic variation to phenotypic co-variation, including epistatic effects of a set of amino-acid substitutions in the reverse transcriptase and protease genes. We show that epistasis increases the pleiotropic degree of single mutations and provides modularity to the GP map of drug resistance in HIV-1. Moreover, modules of epistatic pleiotropic effects within the GP map match the phenotypicmodules of correlated replicative capacity among drug classes. Epistasis thus increases the evolvability of cross-resistance in HIV by providing more drug- and class-specific pleiotropic profiles to the main effects of the mutations. We discuss the implications for the evolution of cross-resistance in HIV. [ABSTRACT FROM AUTHOR]

HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage. [ABSTRACT FROM AUTHOR]

Background. Limited data are available for once-daily (QD) darunavir (DRV)/ritonavir (r) in the pediatric population. Coadministration of etravirine (ETR) may alter the pharmacokinetics (PK) of DRV. We evaluated the PK interactions between DRV/r (QD) and ETR QD or twice-daily (BID) in children, adolescents, and young adults. Methods. Human immunodeficiency virus-infected subjects 9 to < 24 years old on optimized background therapy including DRV/r 800/100 mg QD alone or combined with ETR 200 mg BID or ETR 400 mg QD were enrolled. Protocol-defined target drug exposure ranges based on adult data were used to assess the adequacy of each regimen. Intensive 24-hour blood sampling was performed, and PK parameters were determined using noncompartmental analysis. Results. Thirty-one subjects (14 males) completed the study; 16 received DRV/r QD alone (group 1), 6 received DRV/r plus ETR BID (group 2A), and 9 received DRV/r plus ETR QD (group 2B). The geometric mean (90% confidence interval [CI] geometric mean) for DRV area under the curve at 24 hours (AUC24) was 57.9 (49.6-67.6), 74.9 (44.4-126.5), and 66.4 (50.8-86.9) mg × h/L for patients in groups 1, 2A, and 2B, respectively. The increased DRV exposure when coadministered with ETR was not statistically significant. The geometric mean (90% CI geometric mean) of ETR AUC24 was 8.6 (4.4-16.8) and 11.9 (7.5-18.9) mg × h/L for groups 2A and 2B, respectively, with comparable C24. Conclusions. The results suggest that DRV/r QD with ETR 400 mg QD or 200 mg BID is appropriate and support further evaluation of the safety and efficacy of the once-daily regimen in older children, adolescents, and young adults. [ABSTRACT FROM AUTHOR]

Objectives: This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance. Methods: Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed. Results: Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001). Conclusions: This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby enabling predict the efficacy of pre-clinical drugs for which human pharmacokinetic is not available. [ABSTRACT FROM AUTHOR]

Tenofovir alafenamide (TAF), a novel prodrug of the NtRTI tenofovir (TFV), delivers TFV-diphosphate (TFV-DP) to target cells more efficiently than the current prodrug, tenofovir disoproxil fumarate (TDF), with a 90% reduction in TFV plasma exposure. TAF, within the fixed dose combination of elvitegravir /cobicistat / emtricitabine (FTC)/TAF (E/C/F/TAF), has been evaluated in one Phase 2 and two Phase 3 randomized, double-blinded studies in HIV-infected treatment-naive patients, comparing E/C/F/TAF to E/C/F/TDF. In these studies, the TAF-containing group demonstrated non-inferior efficacy to the TDF-containing comparator group with 91.9% of E/C/F/TAF patients having <50 copies/mL of HIV-1 RNA at week 48. An integrated resistance analysis across these three studies was conducted, including HIV-1 genotypic analysis at screening, and genotypic/phenotypic analysis for patients with HIV-1 RNA >400 copies/mL at virologic failure. Pre-existing primary resistance-associated mutations (RAMs) were observed at screening among the 1903 randomized and treated patients: 7.5% had NRTI-RAMs, 18.2% had NNRTI-RAMs, and 3.4% had primary PI-RAMs. Pre-treatment RAMs did not influence treatment response at Week 48. In the E/C/F/TAF group, resistance development was rare; seven patients (0.7%, 7/978) developed NRTI-RAMs, five of whom (0.5%, 5/978) also developed primary INSTI-RAMs. In the E/C/F/TDF group, resistance development was also rare; seven patients (0.8%, 7/925) developed NRTI-RAMs, four of whom (0.4%, 4/925) also developed primary INSTI-RAMs. An additional analysis by deep sequencing in virologic failures revealed minimal differences compared to population sequencing. Overall, resistance development was rare in E/C/F/TAF-treated patients, and the pattern of emergent mutations was similar to E/C/F/TDF. [ABSTRACT FROM AUTHOR]