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Little is known about the impact of pretreatment drug resistance (PDR) on the efficacy of second generation integrase inhibitors. We sequenced pretreatment plasma specimens from the ADVANCE trial (NCT03122262). Our primary outcome was 96-week virologic success, defined as a sustained viral load <1000 copies/mL from 12 weeks onwards, <200 copies/mL from 24 weeks onwards, and <50 copies/mL after 48 weeks. Here we report how this outcome was impacted by PDR, defined by the World Health Organization (WHO) mutation list. Of 1053 trial participants, 874 (83%) have successful sequencing, including 289 (33%) randomized to EFV-based therapy and 585 (67%) randomized to DTG-based therapy. Fourteen percent (122/874) have ≥1 WHO-defined mutation, of which 98% (120/122) are NNRTI mutations. Rates of virologic suppression are lower in the total cohort among those with PDR 65% (73/112) compared to those without PDR (85% [605/713], P < 0.001), and for those on EFV-based treatment (60% [12/20] vs 86% [214/248], P = 0.002) and for those on DTG-based treatment (61/92 [66%] vs 84% [391/465] P < 0.001, P for interaction by regimen 0.49). Results are similar in multivariable models adjusted for clinical characteristics and adherence. NNRTI resistance prior to treatment is associated with long-term failure of integrase inhibitor-containing first-line regimens, and portends high rates of first-line failure in sub Saharan Africa. Here the authors combine next generation sequencing on plasma from participants of the ADVANCE clinical trial with virological and follow-up data to investigate the impact of pre-treatment drug resistance (PDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) on the efficacy of second-generation integrase inhibitors and find an association between NNRTI resistance prior to treatment and long-term treatment.

In subgroups of two phase 3 studies, patients with high-risk features for failure of antiretroviral therapy, such as a low CD4 count, high base-line level of human immunodeficiency virus (HIV) type 1 RNA, or unfavorable genotypic or phenotypic sensitivity score, raltegravir was superior to placebo in terms of a virologic response at 48 weeks. However, among these patients, in whom antiretroviral therapy had been used previously, 23% of raltegravir recipients had virologic failure by 48 weeks. In patients with high-risk features for failure of antiretroviral therapy, raltegravir was superior to placebo in terms of a virologic response at 48 weeks. However, among these patients, in whom antiretroviral therapy had been used previously, 23% of raltegravir recipients had virologic failure by 48 weeks. Despite the substantial decrease in mortality and morbidity rates associated with highly active antiretroviral therapy over the past decade, there is still a substantial need for effective antiretroviral drugs for patients infected with resistant human immunodeficiency virus type 1 (HIV-1). 1 , 2 The majority of licensed antiretroviral drugs belong to three classes targeting either the HIV-1 protease or reverse transcriptase, and considerable cross-resistance exists among drugs within each class. 3 , 4 In patients with resistant virus, use of antiretroviral agents from new classes offers considerable potential benefit because of the absence of cross-resistance. 5 – 7 HIV-1 integrase represents a new therapeutic target. 8 , . . .

Integrase Strand Transfer Inhibitors (INSTIs) are currently used as the most effective therapy in the treatment of human immunodeficiency virus (HIV) infections. Raltegravir (RAL) and Elvitegravir (EVG), the first generation of INSTIs used successfully in clinical treatment, are susceptible to the emergence of viral resistance and have a high rate of cross-resistance. To counteract these resistant mutants, second-generation INSTI drugs have been developed: Dolutegravir (DTG), Cabotegravir (CAB), and Bictegravir (BIC). However, HIV is also able to develop resistance mechanisms against the second-generation of INSTIs. This review describes the mode of action of INSTIs and then summarizes and evaluates some typical resistance mutations, such as substitution and insertion mutations. The role of unintegrated viral DNA is also discussed as a new pathway involved in conferring resistance to INSTIs. This allows us to have a more detailed understanding of HIV resistance to these inhibitors, which may contribute to the development of new INSTIs in the future.

We describe the efficacy of L-870812, an inhibitor of HIV-1 and SIV integrase, in rhesus macaques infected with the simian-human immunodeficiency virus (SHIV) 89.6P. When initiated before CD4 cell depletion, L-870812 therapy mediated a sustained suppression of viremia, preserving CD4 levels and permitting the induction of virus-specific cellular immunity. L-870812 was also active in chronic infection; however, the magnitude and durability of the effect varied in conjunction with the pretreatment immune response and viral load. These studies demonstrate integrase inhibitor activity in vivo and suggest that cellular immunity facilitates chemotherapeutic efficacy in retroviral infections.

Experimental evidence suggests that a tetramer of integrase (IN) is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.

Background As use of dolutegravir (DTG) becomes more common in resource limited settings (RLS), the demand for integrase resistance testing is increasing. Affordable methods for genotyping all relevant HIV-1 pol genes (i.e., protease (PR ), reverse transcriptase (RT) and integrase (IN)) are required to guide choice of future antiretroviral therapy (ART). We designed an in-house HIV-1 drug resistance (HIVDR) genotyping method that is affordable and suitable for use in RLS. Methods We obtained remnant plasma samples from CAPRISA 103 study and amplified HIV-1 PR, RT and IN genes, using an innovative PCR assay. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. We genotyped samples by Sanger sequencing and assessed HIVDR mutations using the Stanford HIV drug resistance database. We compared drug resistance mutations with previous genotypes and calculated method cost-estimates. Results From 96 samples processed, we obtained sequence data for 78 (81%), of which 75 (96%) had a least one HIVDR mutation, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, 18/78 (23%) had at least one discordant mutation, but only 2/78 (3%) resulted in different phenotypic predictions that could affect choice of subsequent regimen. All CAPRISA 103 study sequences were HIV-1C as confirmed by phylogenetic analysis. Of the 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method were 100% concordant with EQA panel results. Overall genotyping cost per sample was estimated at ~ US$43–$US49, with a processing time of ~ 2 working days. Conclusions We successfully designed an in-house HIVDR method that is suitable for genotyping HIV-1 PR, RT and IN genes, at an affordable cost and shorter turnaround time. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse. The immune synapse promotes cellular information exchange but the role of biophysical forces in synapse function is unclear. Here, the authors show that B cells exert two types of forces, a centripetal myosin II-driven force and a central actin protrusive force at the site of antigen extraction.

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn 7 -like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn 6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair. A programmable transposase integrates donor DNA at user-defined genomic target sites with high fidelity, revealing a new approach for genetic engineering that obviates the need for DNA double-strand breaks and homologous recombination. 

Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Results Here we describe Easi- CRISPR ( E fficient a dditions with s sDNA i nserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi -CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Conclusions Easi- CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi- CRISPR offers a comprehensive means of building large-scale Cre- LoxP animal resources.

AAV vectors that efficiently transduce the mouse brain after intravenous injection are generated with a CRE-dependent selection system. Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer 1 , 2 , 3 , 4 , 5 , 6 . However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics 7 , 8 , 9 , 10 , 11 , 12 , 13 . Here we describe a capsid selection method, called Cre recombination–based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo . We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14 , 15 , 16 , 17 ), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro , it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.