Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir

<p>Abstract</p> <p>Background</p> <p>Human Immunodeficiency Virus type 2 is naturally resistant to some antiretroviral drugs, restricting therapeutic options for patients infected with HIV-2. Regimens including integrase inhibitors (INI) seem to be effective, but little data on HIV-2 integrase (IN) polymorphisms and resistance pathways are available.</p> <p>Materials and methods</p> <p>The <it>integrase </it>coding sequence from 45 HIV-2-infected, INI-naïve, patients was sequenced and aligned against the ROD (group A) or EHO (group B) reference strains and polymorphic or conserved positions were analyzed.</p> <p>To select for raltegravir (RAL)-resistant variants <it>in vitro</it>, the ROD strain was cultured under increasing sub-optimal RAL concentrations for successive rounds. The phenotype of the selected variants was assessed using an MTT assay.</p> <p>Results</p> <p>We describe <it>integrase </it>gene polymorphisms in HIV-2 clinical isolates from 45 patients. Sixty-seven percent of the integrase residues were conserved. The HHCC Zinc coordination motif, the catalytic triad DDE motif, and AA involved in IN-DNA binding and correct positioning were highly conserved and unchanged with respect to HIV-1 whereas the connecting residues of the N-terminal domain, the dimer interface and C-terminal LEDGF binding domain were highly conserved but differed from HIV-1. The N155 H INI resistance-associated mutation (RAM) was detected in the virus population from one ARV-treated, INI-naïve patient, and the 72I and 201I polymorphisms were detected in samples from 36 and 38 patients respectively. No other known INI RAM was detected.</p> <p>Under RAL selective pressure <it>in vitro</it>, a ROD variant carrying the Q91R+I175M mutations was selected. The Q91R and I175M mutations emerged simultaneously and conferred phenotypic resistance (13-fold increase in IC₅₀). The Q91R+I175M combination was absent from all clinical isolates. Three-dimensional modeling indicated that residue 91 lies on the enzyme surface, at the entry of a pocket containing the DDE catalytic triad and that adding a positive charge (Gln to Arg) might compromise IN-RAL affinity.</p> <p>Conclusions</p> <p>HIV-2 polymorphisms from 45 INI-naïve patients are described. Conserved regions as well as frequencies of HIV-2 IN polymorphisms were comparable to HIV-1. Two new mutations (Q91R and I175M) that conferred high resistance to RAL were selected <it>in vitro</it>, which might affect therapeutic outcome.</p

Characteristics and spread to the native population of HIV-1 non-B subtypes in two European countries with high migration rate

Background: Non-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete. Methods: Pol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering. Results: The majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM. Conclusions: The extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero-as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored

Validation of a SARS-CoV-2 Surrogate Neutralization Test Detecting Neutralizing Antibodies against the Major Variants of Concern.

peer reviewedSARS-CoV-2 infection and/or vaccination elicit a broad range of neutralizing antibody responses against the different variants of concern (VOC). We established a new variant-adapted surrogate virus neutralization test (sVNT) and assessed the neutralization activity against the ancestral B.1 (WT) and VOC Delta, Omicron BA.1, BA.2, and BA.5. Analytical performances were compared against the respective VOC to the reference virus neutralization test (VNT) and two CE-IVD labeled kits using three different cohorts collected during the COVID-19 waves. Correlation analyses showed moderate to strong correlation for Omicron sub-variants (Spearman's r = 0.7081 for BA.1, r = 0.7205 for BA.2, and r = 0.6042 for BA.5), and for WT (r = 0.8458) and Delta-sVNT (r = 0.8158), respectively. Comparison of the WT-sVNT performance with two CE-IVD kits, the "Icosagen SARS-CoV-2 Neutralizing Antibody ELISA kit" and the "Genscript cPass, kit" revealed an overall good correlation ranging from 0.8673 to -0.8773 and a midway profile between both commercial kits with 87.76% sensitivity and 90.48% clinical specificity. The BA.2-sVNT performance was similar to the BA.2 Genscript test. Finally, a correlation analysis revealed a strong association (r = 0.8583) between BA.5-sVNT and VNT sVNT using a double-vaccinated cohort (n = 100) and an Omicron-breakthrough infection cohort (n = 91). In conclusion, the sVNT allows for the efficient prediction of immune protection against the various VOCs

Impact of the HIV-1 env Genetic Context outside HR1–HR2 on Resistance to the Fusion Inhibitor Enfuvirtide and Viral Infectivity in Clinical Isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1–HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1–HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1–HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1–HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy

Closing the Ring: A Fourth Extracellular Loop in Chemokine Receptors

Chemokine receptors are heterotrimeric guanine nucleotide binding protein (G protein) coupled receptors (GPCR) that play fundamental roles in many physio- logical and pathological processes. Typically, these receptors form a seven-trans- membrane helix bundle, which is stabilized by a disulfi de bond bridging the top of the third transmembrane segment (TM3) and the second extracellular loop (ECL2). Resolution of the three-dimensional structures of the chemokine receptors CXCR1, CXCR4, and CCR5 revealed the existence of a second disulfi de bridge that links the N terminus of the receptor to the top of the seventh transmembrane segment (TM7), thereby closing the receptor into a ring. An important consequence of this second disulfi de bond is the formation of an additional extracellular loop, which shapes the entrance of the ligand-binding pocket and adds rigidity to the overall surface of the receptor. Here, we discuss the features of these pseudo-loops, the structural re- quirements for their formation, and the effects they may have on receptor functio

Selective-Advantage Profile of Human Immunodeficiency Virus Type 1 Integrase Mutants Explains In Vivo Evolution of Raltegravir Resistance Genotypes▿

The emergence of human immunodeficiency virus type 1 resistance to raltegravir, an integrase strand transfer inhibitor, follows distinct and independent genetic pathways, among which the N155H and Q148HKR pathways are the most frequently encountered in treated patients. After prolonged viral escape, mutants of the N155H pathway are replaced by mutants of the Q148HKR pathway. We have examined the mechanisms driving this evolutionary pattern using an approach that assesses the selective advantage of site-directed mutant viruses as a function of drug concentration. These selective-advantage curves revealed that among single mutants, N155H had the highest and the widest (1 to 500 nM) selective-advantage profile. Despite the higher 50% inhibitory concentration, Q148H displayed a lower and narrower (10 to 100 nM) selective-advantage profile. Among double mutants, the highest and widest selective-advantage profile was seen with G140S+Q148H. This finding likely explains why N155H can be selected early in the course of RAL resistance evolution in vivo but is later replaced by genotypes that include Q148HKR

Additional file 2: Figure S2. of Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation

NF-κB induction relative to Env and CD8-EnvCD expression levels. A. NF-κB induction by subtype B and subtype C Envs relative to Env expression levels. NF-κB induction measured in HEK cells co-transfected with the subtype B or subtype C Envs, NF-κB-Luciferase and CMV-Renilla-Luciferase vectors (Fig. 1a and b) was normalized to Env expression levels (MFI, Additional file 1: Figure S1A) to account for differences in Env expression vectors. B. NF-κB induction by CD8-EnvCD relative to expression levels. NF-κB induction measured in HEK cells co-transfected with the CD8-EnvCD constructs, NF-κB-Luciferase and CMV-Renilla-Luciferase vectors (Fig. 2a and b) was normalized to CD8-EnvCD expression levels (MFI, Additional file 1: Figure S1B) to account for differences in expression vectors. It is noteworthy that this second normalization round is subject to differences in antibody affinity for Env, in Env expression kinetics and cycling dynamics, as well as in Env-induced cytotoxicity. This is particularly the case for the subtype B and C primary Envs, while CD8-EnvCD expression levels are less subject to differences in antibody affinity. (PDF 308 kb

Additional file 1: Figure S1. of Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation

Expression of Env and CD8-EnvCD 37 and 48 h post-transfection by Flow Cytometry. A. Expression of subtype B and C Env in HEK293T cells. 1.2 × 105 HEK293T cells in duplicate wells were cotransfected in the same conditions as in Fig. 1a with all Env expression vectors and the Luciferase expression vectors. The empty pcDNA3.1 vector was used as negative control (mock). Duplicate wells were pooled and Env expression was measured by flow cytometry 37 and 48 h post transfection using a 1:1 mixture of human anti-gp120 antibodies PGT121 + F105 (AIDS Research and Reagent program) and an APC-labelled mouse anti-human IgG secondary antibody (Lifetech A21445). Analyses were performed using FlowJo v10. The mean MFI of at least 3 independent experiments are reported. Error bars represent standard deviation. B. Expression of reference Env and CD8-EnvCD chimeras in HEK293T cells. 1.2 × 105 HEK293T cells in duplicate wells were cotransfected in the same conditions as in Fig. 2a with Env and CD8-EnvCD expression vectors and the luciferase expressing vectors. The empty pcDNA3.1 vector was used as negative control (mock). Duplicate wells were pooled and cells were stained either with the same 1:1 mixture of human anti-gp120 antibodies PGT121 + F105 and an APC-labelled mouse anti-human IgG secondary antibody or with a 510-labelled mouse anti-human CD8 antibody (Biolegend #301048). Analyses were performed using FlowJo v10. The mean MFI of at least 3 independent experiments are reported. Error bars represent standard deviation. (PDF 302 kb