Prevalence and Phenotypic Susceptibility to Doravirine of the HIV-1 Reverse Transcriptase V106I Polymorphism in B and Non-B Subtypes.

BACKGROUND: Limited data are available regarding the susceptibility of the reverse transcriptase V106 polymorphism to doravirine. METHODS: Doravirine susceptibility was measured in site-directed mutants (SDMs) containing V106I, V106A, V106M, and Y188L mutations in subtype B (NL4-3, HXB2) and CRF02_AG background and in recombinant viruses with RT harboring V106I alone derived from 50 people with HIV. RESULTS: HIV-1 B subtype was detected in 1523 of 2705 cases. Prevalence of V106I was 3.2% in B and 2.5% in non-B subtypes, and was higher in subtype F (8.1%) and D (14.3%). Fold-changes (FC) in susceptibility for SDMs were below doravirine biological cutoff (3.0) for V106I, but not for V106A, V106M, and Y188L. Clinically derived viruses tested included 22 B (median FC, 1.2; interquartile range [IQR], 0.9-1.6) and 28 non-B subtypes (median FC, 1.8; IQR, 0.9-3.0). Nine (18%) viruses showed FC values equal or higher than the doravirine biological FC cutoff. CONCLUSIONS: The prevalence of the HIV-1 RT V106I polymorphism in MeditRes HIV consortium remains low, but significantly more prevalent in subtypes D and F. V106I minimally decreased the susceptibility to doravirine in SDMs and most clinical isolates. Reduced susceptibility seems to occur at increased frequency in subtype F1; however, the clinical impact remains to be investigated. CLINICAL TRIALS REGISTRATION: NCT04894357.

Mutations in the 3'-PPT Lead to HIV-1 Replication without Integration.

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 3'-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 3'-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro. This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

Three to four mRNA COVID-19 vaccines in multiple sclerosis patients on immunosuppressive drugs: Seroconversion and variant neutralization.

BACKGROUND AND PURPOSE: An enhanced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine regimen could improve humoral vaccine response in patients with multiple sclerosis (MS) treated by anti-CD20. The aim was to evaluate the serological response and the neutralizing activity after BNT162b2 primary and booster vaccination in MS patients, including patients on anti-CD20 receiving a primary vaccine regimen enhanced with three injections. METHODS: In this prospective longitudinal cohort study of 90 patients (47 on anti-CD20, 10 on fingolimod, 33 on natalizumab, dimethylfumarate or teriflunomide), anti-SARS-CoV-2 receptor binding domain (RBD) immunoglobulin G antibodies were quantified and their neutralization capacity was evaluated by enzyme-linked immunosorbent assay (GenScript) and a virus neutralization test against B.1 historical strain, Delta and Omicron variants, before and after three to four BNT162b2 injections. RESULTS: After the primary vaccination scheme, the anti-RBD positivity rate was strongly decreased in patients on anti-CD20 (28% [15%; 44%] after two shots, 45% [29%; 62%] after three shots) and fingolimod (50% [16%; 84%]) compared to other treatments (100% [90%; 100%]). Neutralization activity was also decreased in patients on anti-CD20 and fingolimod, and notably low for the Omicron variant in all patients (0%-22%). Delayed booster vaccination was performed in 54 patients, leading to a mild increase of anti-RBD seropositivity in patients on anti-CD20 although it was still lower compared to other treatments (65% [43%; 84%] vs. 100% [87%; 100%] respectively). After a booster, Omicron neutralization activity remained low on anti-CD20 and fingolimod treated patients but was strongly increased in patients on other treatments (91% [72%; 99%]). DISCUSSION: In MS patients on anti-CD20, an enhanced primary vaccination scheme moderately increased anti-RBD seropositivity and anti-RBD antibody titre, but neutralization activity remained modest even after a fourth booster injection. TRIAL REGISTRATION INFORMATION: COVIVAC-ID, NCT04844489, first patient included on 20 April 2021.

Neutralization Heterogeneity of UK and South African Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in BNT162b2-Vaccinated or Convalescent Coronavirus Disease 2019 (COVID-19) Healthcare Workers.

There are concerns about neutralizing antibodies' (NAbs') potency against severe acute respiratory syndrome coronavirus 2 variants. Despite decreased NAb titers elicited by BNT162b2 vaccine against VOC202012/01 and 501Y.V2 strains, 28/29 healthcare workers (HCWs) had an NAb titer ≥1:10. In contrast, 6 months after coronavirus disease 2019 mild forms, only 9/15 (60%) of HCWs displayed detectable NAbs against 501Y.V2 strain.

Surveillance of Antibiotic Resistance Genes in Treponema Pallidum Subspecies Pallidum from Patients with Early Syphilis in France.

Benzathine penicillin G (BPG) is the reference treatment for early syphilis, but shortages have recently been reported, highlighting a need for the validation of alternative treatments. The aim of this study was to evaluate the genomic resistance of Treponema pallidum subspecies pallidum (TPA) to macrolides and doxycycline in France. Swabs from genital, anal, oral and cutaneous lesions were obtained from 146 patients with early syphilis in France. They were screened for mutations conferring resistance to macrolides and doxycycline by nested PCR and sequencing. Resistance to macrolides was detected in 85% of the isolates, but no point mutations conferring doxycycline resistance were detected. These findings confirm that, in France, resistance to macrolides is widespread. Moreover, we confirmed the absence of genomic resistance to doxycycline in the TPA strains. Therefore, doxycycline could be safely recommended as an alternative to BPG for the treatment of early syphilis.

Variability of the HIV-1 3' polypurine tract (3'PPT) region and implication in integrase inhibitor resistance.

BACKGROUND: Integrase strand-transfer inhibitors (INSTIs) are efficient at impairing retroviral integration, which is a critical step in HIV-1 replication. To date, resistance to these compounds has been explained by mutations in the viral protein integrase, which catalyses the integration step. Recently, it has been shown that selected mutations in the 3' polypurine tract (3'PPT), a sequence involved in the reverse transcription mechanism, result in high-level resistance to these compounds. This observation was reinforced by the description of a patient who failed INSTI treatment by selecting mutations in the 3'PPT sequence. METHODS: Sequences of the 3'PPT region were analysed in 30706 treatment-naive patients from the public Los Alamos database belonging to six different subtypes and, in parallel, in 107 patients failing INSTI treatment. RESULTS: The analysis showed that the sequences of patients failing INSTI treatment, in the same way as those of treatment-naive patients, are very well conserved regardless of the presence or absence of resistance mutations in the integrase gene. CONCLUSIONS: This study confirms that the selection of a mutation in the 3'PPT region conferring high-level resistance to INSTIs is a rare event. It would require a particular in vivo context and especially a long enough time to be selected, this exposure time being generally reduced by the rapid change of treatment in the case of virological failure. Larger-scale studies in patients with INSTI treatment failure are needed to determine whether the 3'PPT region can play an important role in vivo in INSTI resistance.

Pathway involving the N155H mutation in HIV-1 integrase leads to dolutegravir resistance.

Background: Dolutegravir, an integrase strand-transfer inhibitor (STI), shows a high genetic barrier to resistance. Dolutegravir is reported to be effective against viruses resistant to raltegravir and elvitegravir. In this study, we report the case of a patient treated with dolutegravir monotherapy. Failure of dolutegravir treatment was observed concomitant with the appearance of N155H-K211R-E212T mutations in the integrase (IN) gene in addition to the polymorphic K156N mutation that was present at baseline in this patient. Methods: The impact of N155H-K156N-K211R-E212T mutations was studied in cell-free, culture-based assays and by molecular modelling. Results: Cell-free and culture-based assays confirm that selected mutations in the patient, in the context of the polymorphic mutation K156N present at the baseline, lead to high resistance to dolutegravir requiring that the analysis be done at timepoints longer than usual to properly reveal the results. Interestingly, the association of only N155H and K156N is sufficient for significant resistance to dolutegravir. Modelling studies showed that dolutegravir is less stable in IN/DNA complexes with respect to the WT sequence. Conclusions: Our results indicate that the stability of STI IN/DNA complexes is an important parameter that must be taken into account when evaluating dolutegravir resistance. This study confirms that a pathway including N155H can be selected in patients treated with dolutegravir with the help of the polymorphic K156N that acts as a secondary mutation that enhances the resistance to dolutegravir.

Phenotypic analysis of HIV-1 E157Q integrase polymorphism and impact on virological outcome in patients initiating an integrase inhibitor-based regimen.

Objectives: To assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen. Methods: This was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay. Results: Prevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) <50 copies/mL at initiation and virological suppression was maintained during the first year of follow-up in all but two exhibiting a viral blip. Twenty-four patients had a VL > 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL < 50 copies/mL at week 24 were receiving elvitegravir. The 16 remaining patients were ART experienced in virological failure with drug-resistant viruses displaying several virological outcomes independently of the genotypic susceptibility score. Phenotypic analyses showed a fold change in EC50 of 0.6, 0.9 and 1.9 for raltegravir, dolutegravir and elvitegravir, respectively, in a subtype B context, and 1.1, 1.9 and 2.4 for raltegravir, dolutegravir and elvitegravir, respectively, in a CRF02_AG context. Conclusions: Assessment of virological response in 39 patients initiating an INI-based regimen with E157Q-mutated virus, in combination with phenotypic analysis, suggests that particular attention should be paid to antiretroviral-naive patients and dolutegravir should be preferentially used in these patients.

G118R and F121Y mutations identified in patients failing raltegravir treatment confer dolutegravir resistance.

OBJECTIVES: Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. METHODS: Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. RESULTS: Results obtained with in vitro and ex vivo assays consistently showed that both mutations impaired the catalytic properties of integrase, especially at the integration step. Moreover, both mutations conferred an intermediate level of resistance to dolutegravir. Interestingly, the F121Y mutation, but not the G118R mutation, displayed differential resistance to raltegravir and dolutegravir. Indeed, the F121Y mutation was more resistant to raltegravir than to dolutegravir. CONCLUSIONS: Mutations at G118 and F121, which have been described in patients failing raltegravir-containing regimens, must be included in drug-resistance-testing algorithms.

Combination of two pathways involved in raltegravir resistance confers dolutegravir resistance.

OBJECTIVES: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as raltegravir, elvitegravir or dolutegravir. Three pathways conferring raltegravir/elvitegravir cross-resistance (involving integrase residues Q148, N155 and Y143) were identified. Dolutegravir, belonging to the second generation of strand-transfer compounds, inhibits the Y143 and N155 pathways, but is less efficient at inhibiting the Q148 pathway. The aim of this study was to characterize the combination of two pathways involved in raltegravir resistance described in one patient failing a dolutegravir regimen for their propensity to confer dolutegravir resistance. METHODS: In this study, a patient first failing a regimen including raltegravir was treated with dolutegravir and showed an increase in viruses carrying a combination of two pathways (N155 and Q148). Impacts of these mutations on integrase activity and resistance to strand-transfer inhibitors were characterized using both in vitro and virological assays. RESULTS: Our data showed that the combination of N155H, G140S and Q148H mutations led to strong resistance to dolutegravir. CONCLUSIONS: Combination of N155H, G140S and Q148H mutations originating from two distinct resistance pathways to raltegravir or elvitegravir led to a high level of dolutegravir resistance. Due to its high genetic barrier of resistance, it would be reasonable to use dolutegravir in first-line therapy before emergence of raltegravir or elvitegravir resistance.

Comparative replication capacity of raltegravir-resistant strains and antiviral activity of the new-generation integrase inhibitor dolutegravir in human primary macrophages and lymphocytes.

OBJECTIVES: To evaluate the replication capacity and phenotypic susceptibility to dolutegravir and raltegravir of wild-type and raltegravir-resistant HIV-1 strains in several cellular systems. METHODS: The antiviral activities of dolutegravir and raltegravir were evaluated in human primary monocyte-derived macrophages (MDMs), peripheral blood mononuclear cells (PBMCs) and C8166 T lymphocytic cells. The following raltegravir resistance mutations were analysed: N155H, Y143C, N155H + Y143C and G140S+Q148H. RESULTS: In the absence of drug, the replication capacity of raltegravir-resistant viruses was strongly reduced compared with wild-type in all cellular models analysed. In MDMs and PBMCs, a dramatic decrease in viral replication was observed for the double mutants N155H + Y143C and G140S + Q148H (ranging from 0.1% to 2.5% compared with wild-type). In MDMs, dolutegravir exhibited high potency, with EC50 and EC90 values of 1.1 ± 0.9 and 5.5 ± 3.4 nM, respectively (comparable to raltegravir). These values (particularly for EC90) were significantly lower than those observed in PBMCs (EC50: 2.7 ± 1.5 nM; EC90: 14.8 ± 0.9 nM) and C8166 cells (EC50: 5.5 ± 0.8 nM; EC90: 64.8 ± 5.8 nM). In all cellular models analysed, dolutegravir showed full efficacy against N155H and Y143C mutants (dolutegravir fold-change resistance ranging from 0.1 to 1.4; raltegravir fold-change resistance ranging from 0.1 to 10.3). In C8166 (the only cell model in which replication capacity was sufficient to perform the test) dolutegravir showed full efficacy against mutations N155H + Y143C (dolutegravir fold-change resistance: 0.6) and a slightly lower activity against G140S+Q148H (dolutegravir fold-change resistance: 2.1). CONCLUSIONS: Dolutegravir is effective in different HIV cellular targets and against raltegravir-resistant mutants. The high efficacy of dolutegravir in MDMs (cells with limited metabolism) has relevant clinical implications in light of the role of MDMs in the transmission of HIV infection and dissemination in different body compartments.

New raltegravir resistance pathways induce broad cross-resistance to all currently used integrase inhibitors.

OBJECTIVES: The possibility of replacing raltegravir or elvitegravir with dolutegravir in heavily treatment-experienced patients failing on raltegravir/elvitegravir has been evaluated in VIKING trials. All studied patients failed by the most common pathways, Y143, Q148 and N155, and dolutegravir demonstrated efficacy except for Q148 viruses. The aim of this study was to explore, in the same way, the behaviour of dolutegravir in comparison with raltegravir and elvitegravir against the atypical resistance integrase profiles, G118R and F121Y, described in HIV-1 patients failing on raltegravir therapy. METHODS: The behaviour of integrases with mutations G118R and F121Y towards raltegravir, elvitegravir and dolutegravir was analysed by evaluating phenotypic susceptibility and by means of in silico techniques (investigating binding affinities and the stabilization of the inhibitors in terms of their hydrogen bond network). RESULTS: The phenotypic analysis of G118R and F121Y showed high resistance to raltegravir, elvitegravir and dolutegravir with a fold change >100 when the clinically derived integrase was used, and resistance was also seen when mutations were tested alone in an NL43 backbone, but more often with a lower fold change. In silico, results showed that G118R and F121Y enzymes were associated with reduced binding affinities to each of the inhibitors and with a decreased number of hydrogen bonds compared with the wild-type complexes. CONCLUSIONS: This study showed that G118R and F121Y mutations, rarely described in patients failing on raltegravir, induced broad cross-resistance to all currently used integrase inhibitors. These results are in accordance with our thermodynamic and geometric analysis indicating decreased stability compared with the wild-type complexes.

A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

BACKGROUND: The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. METHODS: Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. RESULTS: Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. CONCLUSIONS: These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

Highly multidrug-resistant HIV: clonal analysis and therapeutic strategies.

OBJECTIVES: There are today HIV-infected patients in therapeutic impasses because of highly multidrug-resistant (HMDR) viruses. We studied the distribution of resistance mutations at clonal level, and we analysed the therapeutic strategies used in such cases to achieve undetectable viraemia. METHODS: The HMDR profile was defined as a genotypic sensitivity score (GSS) ≤ 1.5 for etravirine and raltegravir with full resistance to darunavir. About 30 clones per gene and per patient were sequenced. Virtual phenotypes were determined. Efficacy of therapeutic strategies was evaluated by follow-up of viral loads, CD4 cell counts and trough concentrations of drugs. RESULTS: Among 1310 patients on treatment and with genotypic resistance testing, 25 (2%) were resistant to darunavir and 11 (0.8%) had an HMDR profile. Five-hundred clones could be analysed for four of them. HMDR profiles were harboured by the great majority of clones and all resistance mutations were located on the same strains for all genes. Despite this and a regimen with a GSS <2.0 in three patients, they achieved a viraemia <20 copies/mL. These results were obtained using different strategies: high doses of drugs; combination of antiretrovirals with full or intermediate susceptibility, such as tipranavir, etravirine or maraviroc; and use of alternative compounds, such as foscarnet or interferon. CONCLUSION: Patients with HMDR HIV were uncommon, but, in such cases, all resistance mutations were borne on the same majority strains. In this study, tipranavir was the only protease inhibitor with full or intermediate susceptibility. Despite very limited therapeutic options, an undetectable viraemia can be achieved by combining different strategies.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Corrigendum: XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants.

[This corrects the article DOI: 10.3389/fimmu.2021.761250.].

Golgi localization of SARS-CoV-2 spike protein and interaction with furin in cerebral COVID-19 microangiopathy: a clue to the central nervous system involvement?

In a neuropathological series of 20 COVID-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by MRI. The cases presented with microhemorrhages evocative of small artery diseases. This COVID-19 associated cerebral microangiopathy (CCM) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. There was evidence of blood-brain-barrier leakage. Fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. While no viral particle or viral RNA was found in the brain, the SARS-CoV-2 spike protein was detected in the Golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. Endothelial cells in culture were not permissive to SARS-CoV-2 replication. The distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. In the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. In contrast, in cerebral endothelial cells the excretion cycle was blocked in the Golgi apparatus. Interruption of the excretion cycle could explain the difficulty of SARS-CoV-2 to infect endothelial cells in vitro and to produce viral RNA in the brain. Specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of COVID-19 associated cerebral microangiopathy. Furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy.

HIV-1 genome is often defective in PBMCs and rectal tissues after long-term HAART as a result of APOBEC3 editing and correlates with the size of reservoirs.

OBJECTIVES: Precise characterization of viruses present in reservoirs in long-term pretreated patients will be a major issue to consider in the context of viral eradication. We assessed the frequency of defective viruses present in cellular reservoirs. METHODS: Peripheral blood mononuclear cells (PBMCs) and rectal biopsy samples were compared between five patients on successful long-term highly active antiretroviral therapy (HAART) (>7 years without blips) and five untreated patients. Molecular cloning and sequencing of the reverse transcriptase region were used to detect the presence of and quantify in-frame stop codons in HIV quasi-species. The relationship between the size of the reservoir and the frequency of defective genomes was assessed. RESULTS: Defective genomes were systematically detected in all patients on long-term HAART in both compartments (PBMCs and rectal tissues), with a higher level of defective genomes per sample compared with PBMCs of untreated patients. A high level of defective genomes was correlated with a small size of HIV proviral DNA. Regarding the nucleotide context, guanine (G) to adenine (A) substitution at tryptophan positions was responsible for the appearance of 89% of all in-frame stop codons in the context of G-to-A hypermutation, likely reflecting APOBEC3 footprints on the viral genome. CONCLUSIONS: We propose a scenario whereby defective genomes accumulate during HAART treatment, eventually reaching a viral extinction threshold. In the context of viral eradication, measurement of the relative amounts of defective and non-defective viruses (by molecular cloning and ultradeep sequencing) should be used as a new criterion for eradicating HIV.

Risk factors for raltegravir resistance development in clinical practice.

OBJECTIVES: To investigate the best conditions of raltegravir use to avoid the selection of resistance mutations in the three main genetic pathways: 148, 155 and 143. METHODS: A total of 161 patients failing on raltegravir with two consecutive HIV-1 viral loads >20 copies/mL were studied. Ten parameters [HIV-1 RNA and CD4 at baseline and failure, genotypic sensitivity score (GSS) of treatment associated with raltegravir, protease inhibitors used, time spent on raltegravir, subtype, sex and age] were tested in univariate and multivariate logistic regression analyses and compared with the emergence of resistance mutations to raltegravir at failure. Phenotypic susceptibility to raltegravir was studied in 16 patients without the main resistance mutations to raltegravir at failure. RESULTS: At raltegravir failure, 46/161 patients (28.6%) had integrase resistance mutations, whereas 115/161 (71.4%) had no resistance mutations. High HIV-1 viral load level at failure (OR = 2.81, 95% CI 1.8-4.6, P < 0.001) and low GSS of treatment associated with raltegravir (OR = 11.6, 95% CI 4.5-36.4, P < 0.001) were independently associated with the selection of raltegravir mutations. The percentages of patients with integrase resistance mutations were 7.7% (6/78) versus 48.1% (40/83) when viral load is ≤200 or >200 copies/mL and 47.5% (39/82) versus 8.9% (7/79) when GSS is <2 or ≥2. Among patients without main resistance mutations, two patients showed raltegravir phenotypic resistance, one naturally with F121Y at baseline and the other acquiring G118R at failure. CONCLUSIONS: Our results show that to avoid the selection of raltegravir resistance mutations, patients have to be treated with at least two active drugs in combination with raltegravir and to maintain a viral load ≤200 copies/mL.

SARS-CoV-2 Neutralizing Responses in Various Populations, at the Time of SARS-CoV-2 Variant Virus Emergence: Evaluation of Two Surrogate Neutralization Assays in Front of Whole Virus Neutralization Test.

The SARS-CoV-2 neutralizing antibodies response is the best indicator of effective protection after infection and/or vaccination, but its evaluation requires tedious cell-based experiments using an infectious virus. We analyzed, in 105 patients with various histories of SARS-CoV-2 infection and/or vaccination, the neutralizing response using a virus neutralization test (VNT) against B.1, Alpha, Beta and Omicron variants, and compared the results with two surrogate assays based on antibody-mediated blockage of the ACE2-RBD interaction (Lateral Flow Boditech and ELISA Genscript). The strongest response was observed for recovered COVID-19 patients receiving one vaccine dose. Naïve patients receiving 2 doses of mRNA vaccine also demonstrate high neutralization titers against B.1, Alpha and Beta variants, but only 34.3% displayed a neutralization activity against the Omicron variant. On the other hand, non-infected patients with half vaccination schedules displayed a weak and inconstant activity against all isolates. Non-vaccinated COVID-19 patients kept a neutralizing activity against B.1 and Alpha up to 12 months after recovery but a decreased activity against Beta and Omicron. Both surrogate assays displayed a good correlation with the VNT. However, an adaptation of the cut-off positivity was necessary, especially for the most resistant Beta and Omicron variants. We validated two simple and reliable surrogate neutralization assays, which may favorably replace cell-based methods, allowing functional analysis on a larger scale.