HIV-1 genotypic resistance testing using single molecule real-time sequencing.

BACKGROUND: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories. OBJECTIVES: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping. STUDY DESIGN: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches. RESULTS: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001). CONCLUSIONS: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT- mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy.

BACKGROUND: The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. METHODS: We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. FINDINGS: Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. INTERPRETATION: Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. FUNDING: This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

Evaluation of two anti-SARS-CoV-2 antibody immunoassays for monitoring patients on pre-exposure prophylaxis.

Pre-exposure prophylaxis (PrEP) is crucial to prevent severe COVID-19 in immunocompromised patients. A reliable method is needed to quantify anti-SARS-CoV-2 antibody levels for personalized monitoring during PrEP. We measured the binding antibody concentrations of 63 immunocompromised patients receiving 300mg or 600mg tixagevimab/cilgavimab on PrEP day and twice during the following 3 months. All blood samples were tested using the Abbott anti-SARS-CoV-2 IgG II Quant assay, the Roche Elecsys anti-SARS-CoV-2 S assay, and live virus-based neutralization assays. The results of the two immunoassays were correlated on day 0, 1 month, and 3 months post-PrEP. Passing-Bablok regression demonstrated higher anti-S concentration values measured with the Roche immunoassay compared to those measured with the Abbott immunoassay. Antibody concentrations were higher after 600 mg tixagevimab/cilgavimab prophylaxis than after 300 mg. The neutralizing antibody titers obtained using the omicron BA.5 and BA.2.75 strains were low. Both automated immunoassays are suitable for monitoring immunocompromised patients on PrEP.

Impact of Casirivimab-Imdevimab on Severe Acute Respiratory Syndrome Coronavirus 2 Delta Variant Nasopharyngeal Virus Load and Spike Quasispecies.

Background: The increasing use of monoclonal antibodies (mAbs) to treat coronavirus disease 2019 raises questions about their impact on the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mAb-resistant variants. We assessed the impact of Casirivimab-Imdevimab on SARS-CoV-2 mutations associated with reduced mAb activity in treated patients. Methods: We measured the nasopharyngeal (NP) viral load and sequenced the haplotypes of spike gene of 50 patients infected with the SARS-CoV-2 delta variant and treated with Casirivimab-Imdevimab using single-molecule real-time sequencing. Results: The NP SARS-CoV-2 viral load of patients treated with Casirivimab-Imdevimab decreased from 8.13 (interquartile range [IQR], 7.06-8.59) log10 copies/mL pretreatment to 3.67 (IQR, 3.07-5.15) log10 copies/mL 7 days later (P < .001). Of the 36 patients for whom follow-up timepoints Spike sequencing were available, none of the Spike mutations that reduced mAb activity were detected. Conclusions: Casirivimab-Imdevimab is an effective treatment for patients infected with the SARS-CoV-2 delta variant. Despite selective pressure on SARS-CoV-2 Spike quasispecies, we detected no key mutations that reduced mAb activity in our patients.

Influence of treatment with neutralizing monoclonal antibodies on the SARS-CoV-2 nasopharyngeal load and quasispecies.

OBJECTIVES: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness. PATIENTS AND METHODS: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients). RESULTS: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients. CONCLUSION: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.

Testing individual and pooled saliva samples for sars-cov-2 nucleic acid: a prospective study.

Control of the rapid spread of the SARS-CoV-2 virus requires efficient testing. We collected paired nasopharyngeal swab (NPs) and saliva samples from 303 subjects (52.8% symptomatic) at a drive-through testing center; 18% of whom tested positive. The NPs, salivas and five saliva pools were tested for SARS-CoV-2 RNA using the Aptima™ assay and a laboratory-developed test (LDT) on the Panther-Fusion™ Hologic® platform. The saliva sensitivity was 80% (LDT) and 87.5% (Aptima™) whereas that of NPs was 96.4% in both assays. The pooled saliva sensitivity of 72.7% (LDT) and 75% (Aptima™) was not significantly different of that of individual saliva testing. Saliva specimens appear to be suitable for sensitive non-invasive assays to detect SARS-CoV-2 nucleic acid; pooling them for a single test will improve laboratory throughput.