Does social need fulfillment moderate the association between socioeconomic status and health risk behaviours during pregnancy?

Socioeconomic differences in health risk behaviours during pregnancy may be influenced by social relations. In this study, we aimed to investigate if social need fulfillment moderates the association between socioeconomic status (SES) and health risk behaviours (smoking and/or alcohol consumption) during pregnancy. We used baseline data from the Lifelines Cohort Study merged with data from the Lifelines Reproductive Origin of Adult Health and Disease (ROAHD) cohort. Education level was used to determine SES, categorized into low, middle, and high, with middle SES as the reference category. Social need fulfillment was taken as indicator for social relations and was measured with the validated Social Production Function Instrument for the Level of Well-being scale. The dependent variable was smoking and/or alcohol consumption during pregnancy. Univariable and multivariable logistic regression analysis was conducted to assess the association of SES and social need fulfillment with health risk behaviours and to test for effect modification. We included 1107 pregnant women. The results showed that women with a high SES had statistically significantly lower odds of health risk behaviours during pregnancy. The interaction effect between SES and social need fulfillment on health risk behaviours was not statistically significant, indicating that no moderation effect is present. The results indicate that social need fulfillment does not modify the effect of SES on health risk behaviours during pregnancy. However, in literature, social relations are identified as an important influence on health risk behaviours. More research is needed to identify which measure of social relations is the most relevant regarding the association with health risk behaviours.

Rapid Rebound of a Preexisting CXCR4-tropic Human Immunodeficiency Virus Variant After Allogeneic Transplantation With CCR5 Δ32 Homozygous Stem Cells.

Allogeneic stem cell transplantation (alloSCT) of homozygous CCR5 Δ32 stem cells once resulted in the cure of human immunodeficiency virus (HIV) infection. We have recently reported a viral breakthrough in a similar setting. Here, we demonstrate that the rapid rebound after alloSCT was related to a highly replicative CXCR4-tropic HIV variant, which could already be detected before alloSCT.

GS-8374, a prototype phosphonate-containing inhibitor of HIV-1 protease, effectively inhibits protease mutants with amino acid insertions.

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.

Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer.

Nucleotide-competing reverse transcriptase inhibitors were shown to bind reversibly to the nucleotide-binding site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). Here, we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R, and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analog phosphonoformic acid. However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on these data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity.

BACKGROUND: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. RESULTS: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. CONCLUSIONS: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Dynamics of Low-Level Viremia and Immune Activation after Switching to a Darunavir-Based Regimen.

There is an ongoing debate regarding whether low-level viremia (LLV), in particular persistent LLV, during HIV treatment with optimal adherence originates from low-level viral replication, viral production, or both. We performed an observational study in 30 individuals with LLV who switched to a boosted darunavir (DRV)-based therapy. In-depth virological analyses were used to characterize the viral population and the (activity) of the viral reservoir. Immune activation was examined using cell-bound and soluble markers. The primary outcome was defined as the effect on HIV-RNA and was categorized by responders (<50 cp/mL) or non-responders (>50 cp/mL). At week 24, 53% of the individuals were considered responders, 40% non-responders, and 7% could not be assigned. Sequencing showed no evolution or selection of drug resistance in the non-responders. Production of defective virus with mutations in either the protease (D25N) or RT active site contributed to persistent LLV in two individuals. We show that in about half of the study participants, the switch to a DRV-based regimen resulted in a viral response indicative of ongoing low-level viral replication as the cause of LLV before the switch. Our data confirm that in clinical management, high genetic barrier drugs like DRV are a safe choice, irrespective of the source of LLV.

Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity.

BACKGROUND: Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V). RESULTS: Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one.The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant. CONCLUSIONS: The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.

HIV-1 pretreatment drug resistance negatively impacts outcomes of first-line antiretroviral treatment.

INTRODUCTION: Pretreatment drug resistance (PDR) prevalence in sub-Saharan Africa is rising, but evidence of its impact on efavirenz (EFV)-based antiretroviral treatment (ART) is inconclusive. We determined the impact of PDR on outcomes of EFV-based ART in a subanalysis of a randomized clinical trial comparing different ART monitoring strategies implemented at a rural treatment facility in Limpopo, South Africa. METHODS: Participants initiating EFV-based first-line ART (2015-2017) were enrolled and received 96 weeks follow-up. Resistance to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and non-NRTI's (NNRTIs) was retrospectively assessed by population-based sequencing. Virological failure was defined as a viral load of at least 1000 copies/ml after at least 24 weeks of ART. RESULTS: A total of 207 participants were included, 60.4% (125/207) of whom were female. Median age was 38.8 (interquartile range: 31.4-46.7) years. Median CD4+ cell count was 191 (interquartile range: 70-355) cells/µl. PDR was detected in 12.9% (25/194) of participants with available sequencing results; 19 had NNRTI-resistance, and six had NRTI- and NNRTI-resistance. 26.0% of participants (40/154) with sequencing results and virological follow-up developed virological failure. PDR was independently associated with failure (adjusted hazard ratio: 3.7 [95% confidence interval: 1.68.5], P = 0.002). At failure, 87.5% (7/8) of participants with PDR harboured dual-class resistant virus, versus 16.7% (4/24) of participants without PDR (P = 0.0007). Virological resuppression after failure on first-line ART occurred in 57.7% (15/26) of participants without PDR versus 14.3% (1/7) of participants with PDR (P = 0.09). CONCLUSION: PDR was detected in 13% of study participants. PDR significantly increased the risk of virological failure of EFV-based ART. Accumulation of resistance at failure and inability to achieve virological resuppression illustrates the profound impact of PDR on treatment outcomes.

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies.

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.

A randomized study of intensified antiretroviral treatment monitoring versus standard-of-care for prevention of drug resistance and antiretroviral treatment switch.

INTRODUCTION: Standard-of-care antiretroviral treatment (ART) monitoring in low and middle-income countries consists of annual determination of HIV-RNA viral load with confirmatory viral load testing in case of viral rebound. We evaluated an intensified monitoring strategy of three-monthly viral load testing with additional drug exposure and drug resistance testing in case of viral rebound. METHODS: We performed an open-label randomized controlled trial (RCT) at a rural South African healthcare clinic, enrolling adults already receiving or newly initiating first-line ART. During 96 weeks follow-up, intervention participants received three-monthly viral load testing and sequential point-of-care drug exposure testing and DBS-based drug resistance testing in case of rebound above 1000 copies/ml. Control participants received standard-of-care monitoring according to the WHO guidelines. RESULTS: Five hundred one participants were included, of whom 416 (83.0%) were randomized at 24 weeks. Four hundred one participants were available for intention-to-treat analysis. Viral rebound occurred in 9.0% (18/199) of intervention participants and in 11.9% (24/202) of controls ( P  = 0.445). Time to detection of rebound was 375 days [interquartile range (IQR): 348-515] in intervention participants and 360 days [IQR: 338-464] in controls [hazard ratio: 0.88 (95% confidence interval (95% CI): 0.46-1.66]; P  = 0.683]. Duration of viral rebound was 87 days [IQR: 70-110] in intervention participants and 101 days [IQR: 78-213] in controls ( P  = 0.423). In the control arm, three patients with confirmed failure were switched to second-line ART. In the intervention arm, of three patients with confirmed failure, switch could initially be avoided in two cases. CONCLUSION: Three-monthly viral load testing did not significantly reduce the duration of viraemia when compared with standard-of-care annual viral load testing, providing randomized trial evidence in support of annual viral load monitoring.

In-depth Characterization of Vaccine Breakthrough Infections With SARS-CoV-2 Among Health Care Workers in a Dutch Academic Medical Center.

Severe acute respiratory syndrome coronavirus 2 infection after coronavirus disease 2019 vaccination raises concerns about the emergence of vaccine escape variants. Here we characterize 14 breakthrough infections among 5860 fully vaccinated Dutch health care workers ≥14 days after the final dose of vaccination with either BNT162b2, mRNA-1273, or Ad26.COV2.S. These breakthrough infections presented with regular B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants and high viral loads, despite normal vaccine-induced B- and T-cell immune responses detected by live virus neutralization assays and ELISpot. High-risk exposure settings, such as in households, indicate a potential risk of viral transmission despite full vaccination.

Autopsy Study Defines Composition and Dynamics of the HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell Transplantation with CCR5Δ32/Δ32 Donor Cells.

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

Maraviroc is able to inhibit dual-R5 viruses in a dual/mixed HIV-1-infected patient.

OBJECTIVES: Maraviroc is the first licensed chemokine co-receptor 5 (CCR5) co-receptor antagonist in clinical practice. It is currently being used in patients harbouring exclusively CCR5-tropic virus. The objective of the study was to investigate the impact of maraviroc on viruses with different co-receptor preferences in a patient with a dual/mixed (D/M) infection. METHODS: We present a case report of an HIV-1 patient infected with a D/M virus population. Co-receptor tropism was determined by phenotypic and genotypic tests. Biological clones from pre- and post-maraviroc therapy were generated. Tropism of these infectious clones was investigated in U373-MAGI cells expressing CD4+ CCR5+ or CD4+ CXCR4+. Maraviroc susceptibility and viral replication were determined using donor peripheral blood mononuclear cells (PBMCs). RESULTS: In-depth clonal genotypic analysis revealed the presence of both R5-tropic variants and X4-tropic viruses before the start of maraviroc. During maraviroc therapy all R5-predicted viruses were suppressed. Phenotypic analyses revealed that all biological clones before maraviroc therapy could infect both CCR5- and CXCR4-bearing U373-MAGI cells, demonstrating dual tropism. The baseline biological clones preferentially infected the CCR5 cell line and were fully susceptible to maraviroc in PBMCs (dual-R5). In contrast, during maraviroc therapy the dual-R5-tropic viruses were replaced by more X4-tropic viruses (dual-X4), which could not be inhibited by maraviroc. CONCLUSIONS: This case report demonstrates that dual-tropic viruses, capable of using both co-receptors in phenotypic assays, can be inhibited by maraviroc if they have a CCR5 co-receptor preference in vivo.

Immune activation correlates with and predicts CXCR4 co-receptor tropism switch in HIV-1 infection.

HIV-1 cell entry is mediated by binding to the CD4-receptor and chemokine co-receptors CCR5 (R5) or CXCR4 (X4). R5-tropic viruses are predominantly detected during early infection. A switch to X4-tropism often occurs during the course of infection. X4-tropism switching is strongly associated with accelerated disease progression and jeopardizes CCR5-based HIV-1 cure strategies. It is unclear whether host immunological factors play a causative role in tropism switching. We investigated the relationship between immunological factors and X4-tropism in a cross-sectional study in HIV-1 subtype C (HIV-1C)-infected patients and in a longitudinal HIV-1 subtype B (HIV-1B) seroconverter cohort. Principal component analysis identified a cluster of immunological markers (%HLA-DR+ CD4+ T-cells, %CD38+HLA-DR+ CD4+ T-cells, %CD38+HLA-DR+ CD8+ T-cells, %CD70+ CD4+ T-cells, %CD169+ monocytes, and absolute CD4+ T-cell count) in HIV-1C patients that was independently associated with X4-tropism (aOR 1.044, 95% CI 1.003-1.087, p = 0.0392). Analysis of individual cluster contributors revealed strong correlations of two markers of T-cell activation (%HLA-DR+ CD4+ T-cells, %HLA-DR+CD38+ CD4+ T-cells) with X4-tropism, both in HIV-1C patients (p = 0.01;p = 0.03) and HIV-1B patients (p = 0.0003;p = 0.0001). Follow-up data from HIV-1B patients subsequently revealed that T-cell activation precedes and independently predicts X4-tropism switching (aHR 1.186, 95% CI 1.065-1.321, p = 0.002), providing novel insights into HIV-1 pathogenesis and CCR5-based curative strategies.

Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region.

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms.

INTRODUCTION: The latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype-tolerance using digital PCR. METHODS: A subtype-B-specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype-tolerance in digital PCR (Bio-Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide. RESULTS: HIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty-nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG. CONCLUSIONS: The results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non-B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

A novel substrate-based HIV-1 protease inhibitor drug resistance mechanism.

BACKGROUND: HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. METHODS AND FINDINGS: We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. CONCLUSIONS: HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape.

HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.

Short Communication: In Vitro Accumulation of Drug Resistance Mutations in Chimeric Infectious Clones Containing Subtype B or C Reverse Transcriptase and Selected with Tenofovir or Didanosine.

Highly active antiretroviral therapy (HAART) contributed to the improvement in the life expectancy of HIV-infected patients. However, the emergence of drug-resistant mutations (DRM) is a major viral factor impacting therapeutic failure. Differences in DRM can occur among HIV-1 subtypes. We evaluate the kinetics of the selection of resistance mutations in vitro analyzing two chimeric clones that contain the reverse transcriptases of subtypes B or C (RTB' and RTC') in cells treated with increasing concentrations of tenofovir disoproxil fumarate (TDF) and didanosine (ddI). The mutation K65R is selected more quickly in RTC' than in RTB' viruses with TDF and ddI, and additional mutations (positions 45, 62, and 68) were selected after K65R fixation. Other primary mutations (M184V and Q151M) were selected with ddI treatment in conjunction with K65R only in RTC' viruses. Both patterns, M184V+K65R and Q151M+K65R, have a significant impact on NRTI resistance. Our data suggest that selection of TDF and ddI DRMs can occur earlier in subtype C HIV in patients when compared to subtype B.

Differential in vitro kinetics of drug resistance mutation acquisition in HIV-1 RT of subtypes B and C.

BACKGROUND: HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC). METHODS/PRINCIPAL FINDINGS: MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F). CONCLUSIONS/SIGNIFICANCE: This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.