Upper Airway Epithelial Tissue Transcriptome Analysis Reveals Immune Signatures Associated with COVID-19 Severity in Ghanaians.

The immunological signatures driving the severity of coronavirus disease 19 (COVID-19) in Ghanaians remain poorly understood. We performed bulk transcriptome sequencing of nasopharyngeal samples from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected Ghanaians with mild and severe COVID-19, as well as healthy controls to characterize immune signatures at the primary SARS-CoV-2 infection site and identify drivers of disease severity. Generally, a heightened antiviral response was observed in SARS-CoV-2-infected Ghanaians compared with uninfected controls. COVID-19 severity was associated with immune suppression, overexpression of proinflammatory cytokines, including CRNN, IL1A, S100A7, and IL23A, and activation of pathways involved in keratinocyte proliferation. SAMD9L was among the differentially regulated interferon-stimulated genes in our mild and severe disease cohorts, suggesting that it may play a critical role in SARS-CoV-2 pathogenesis. By comparing our data with a publicly available dataset from a non-African (Indians) (GSE166530), an elevated expression of antiviral response-related genes was noted in COVID-19-infected Ghanaians. Overall, the study describes immune signatures driving COVID-19 severity in Ghanaians and identifies immune drivers that could serve as potential prognostic markers for future outbreaks or pandemics. It further provides important preliminary evidence suggesting differences in antiviral response at the upper respiratory interface in sub-Saharan Africans (Ghanaians) and non-Africans, which could be contributing to the differences in disease outcomes. Further studies using larger datasets from different populations will expand on these findings.

Recommendations for empowering early career researchers to improve research culture and practice.

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.

Detection of SARS-CoV-2 intra-host recombination during superinfection with Alpha and Epsilon variants in New York City.

Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, detection of recombination is only feasible when genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was infected with two distinct viral variants: Alpha (B.1.1.7) and Epsilon (B.1.429). This superinfection was first noted when an Alpha genome sequence failed to exhibit the classic S gene target failure behavior used to track this variant. Full genome sequencing from four independent extracts reveals that Alpha variant alleles comprise around 75% of the genomes, whereas the Epsilon variant alleles comprise around 20% of the sample. Further investigation reveals the presence of numerous recombinant haplotypes spanning the genome, specifically in the spike, nucleocapsid, and ORF 8 coding regions. These findings support the potential for recombination to reshape SARS-CoV-2 genetic diversity.

A SARS-CoV-2 nucleocapsid ELISA represents a low-cost alternative to lateral flow testing for community screening in LMI countries.

Background Controlling the spread of SARS-CoV-2 is problematic because of transmission driven by asymptomatic and pre-symptomatic individuals. Community screening can help identify these individuals but is often too expensive for countries with limited health care resources. Low-cost ELISA assays may address this problem, but their use has not yet been widely reported. Methods We developed a SARS-CoV-2 nucleocapsid ELISA and assessed its diagnostic performance on nose and throat swab samples from UK hospitalised patients and sputum samples from patients in Ghana. Results The ELISA had a limit of detection of 8.4 pg/ml antigen and 16 pfu/ml virus. When tested on UK samples (128 positive and 10 negative patients), sensitivity was 58.6% (49.6-67.2) rising to 78.3% (66.7-87.3) if real-time PCR Ct values > 30 were excluded, while specificity was 100% (69.2-100). In a second trial using the Ghanaian samples (121 positive, 96 negative), sensitivity was 52% (42.8-61.2) rising to 72.6% (61.8-81.2) when a > 30 Ct cut-off was applied, while specificity was 100% (96.2-100). Conclusions: Our data show that nucleocapsid ELISAs can test a variety of patient sample types while achieving levels of sensitivity and specificity required for effective community screening. Further investigations into the opportunities that this provides are warranted.

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana.

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12-April 1 2020) and 31 from later time-points ( 25-27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5-99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.

Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir.

OBJECTIVES: The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. METHODS: Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. RESULTS: R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. CONCLUSIONS: Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 µM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 µM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

The R263K substitution in HIV-1 subtype C is more deleterious for integrase enzymatic function and viral replication than in subtype B.

OBJECTIVES: Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. DESIGN AND METHODS: We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. RESULTS: Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively - significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. CONCLUSIONS: The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.

Dolutegravir maintains a durable effect against HIV replication in tissue culture even after drug washout.

OBJECTIVES: Of the currently approved HIV integrase strand transfer inhibitors (INSTIs), dolutegravir has shown greater efficacy than raltegravir at suppressing HIV-1 replication in treatment-experienced individuals. Biochemical experiments have also shown that dolutegravir has a longer dissociative half-life when bound to HIV integrase than does raltegravir. In order to study the intracellular efficacy of various INSTIs, we asked whether drug removal from INSTI-treated HIV-1-infected cells would result in different times to viral rebound. In addition, we assessed the role of the R263K substitution within the integrase ORF that is associated with low-level resistance to dolutegravir. METHODS: HIV-infected MT-2 cells were treated with dolutegravir, raltegravir or a third experimental INSTI (MK-2048) and the drugs were washed out after varying times. Viral replication was monitored by measuring reverse transcriptase (RT) activity in the culture fluids. RESULTS: We observed a significantly slower increase in RT activity after the removal of dolutegravir compared with raltegravir or MK-2048. The incubation time before the drug was removed also had an impact on the level of RT activity independently of the drug and virus used. The R263K substitution did not significantly impact on levels of RT activity after drug washout, suggesting that dolutegravir remained tightly bound to the integrase enzyme despite the presence of this mutation. CONCLUSIONS: These results suggest that the residency time of INSTIs on integrase is a key factor in the activity of these drugs and that the anti-HIV activity of dolutegravir persists more effectively than that of other INSTIs after drug washout.

Structural Studies of the HIV-1 Integrase Protein: Compound Screening and Characterization of a DNA-Binding Inhibitor.

Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance.

A resveratrol analog termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene is a potent HIV-1 inhibitor.

HIV resistance to current anti-HIV drugs and drug toxicity have created a need for new anti-HIV agents. We have examined and characterized a synthetic resveratrol analog, termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (M8), for potential anti-HIV activity. Here, we demonstrate that M8 possesses potent anti-HIV activity against several HIV variants with EC50 values in the low µM range. M8 was shown to act at a very early step of HIV entry prior to fusion to host cells. These results demonstrate that this novel resveratrol derivative possesses potent anti-HIV-1 activity and may have a mechanism of action that is different from current anti-HIV-1 drugs including entry inhibitors. Further structure-guided design might lead to the development of newer improved resveratrol derivatives that could have value either in therapy or as microbicides to prevent the sexual transmission of HIV-1.

Subtype-specific analysis of the K65R substitution in HIV-1 that confers hypersusceptibility to a novel nucleotide-competing reverse transcriptase inhibitor.

Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype.

Differential effects of the G118R, H51Y, and E138K resistance substitutions in different subtypes of HIV integrase.

UNLABELLED: Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE: Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3' processing indicates the importance of the 3' processing step and highlights a therapeutic angle that needs to be better exploited.

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity.

BACKGROUND: The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. METHODS: We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. RESULTS: We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. CONCLUSIONS: The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic.

Effect of HIV-1 integrase resistance mutations when introduced into SIVmac239 on susceptibility to integrase strand transfer inhibitors.

UNLABELLED: Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36- and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred high-level resistance to both RAL and EVG (>300- and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs. IMPORTANCE: The goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.

Integrase strand transfer inhibitors in the management of HIV-positive individuals.

The use of highly active antiretroviral therapy against human immunodeficiency virus (HIV) can lead to rare instances of treatment failure and the emergence of drug resistance. HIV drug-resistant strains are archived in cellular reservoirs, and this can exclude the future efficacy of drugs or drug classes against which resistance has emerged. In addition, drug-resistant viruses can be transmitted between individuals. HIV drug resistance has been countered through the constant development of new antiretroviral drugs. Integrase strand transfer inhibitors, that actively block the integration of the HIV genome into the host DNA, represent the most recent antiretroviral drugs. Of these, raltegravir, elvitegravir, and dolutegravir are the only integrase strand transfer inhibitors that have been approved for human therapy by the US Food and Drug Administration. Dolutegravir is unique in its ability to seemingly evade HIV drug resistance in treatment-naïve individuals. Here, we review the use of integrase strand transfer inhibitors in the management of HIV, focusing on HIV resistance.

Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors.

OBJECTIVE: Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. DESIGN AND METHODS: We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. RESULTS: Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. CONCLUSION: Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness.

BACKGROUND: First-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL. RESULTS: Using biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation. CONCLUSIONS: Since the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.