Nirsevimab for Prevention of RSV in Healthy Late-Preterm and Term Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection and hospitalization in infants. Nirsevimab is a monoclonal antibody to the RSV fusion protein that has an extended half-life. The efficacy and safety of nirsevimab in healthy late-preterm and term infants are uncertain. METHODS: We randomly assigned, in a 2:1 ratio, infants who had been born at a gestational age of at least 35 weeks to receive a single intramuscular injection of nirsevimab or placebo before the start of an RSV season. The primary efficacy end point was medically attended RSV-associated lower respiratory tract infection through 150 days after the injection. The secondary efficacy end point was hospitalization for RSV-associated lower respiratory tract infection through 150 days after the injection. RESULTS: A total of 1490 infants underwent randomization: 994 were assigned to the nirsevimab group and 496 to the placebo group. Medically attended RSV-associated lower respiratory tract infection occurred in 12 infants (1.2%) in the nirsevimab group and in 25 infants (5.0%) in the placebo group; these findings correspond to an efficacy of 74.5% (95% confidence interval [CI], 49.6 to 87.1; P<0.001) for nirsevimab. Hospitalization for RSV-associated lower respiratory tract infection occurred in 6 infants (0.6%) in the nirsevimab group and in 8 infants (1.6%) in the placebo group (efficacy, 62.1%; 95% CI, -8.6 to 86.8; P = 0.07). Among infants with data available to day 361, antidrug antibodies after baseline were detected in 58 of 951 (6.1%) in the nirsevimab group and in 5 of 473 (1.1%) in the placebo group. Serious adverse events were reported in 67 of 987 infants (6.8%) who received nirsevimab and in 36 of 491 infants (7.3%) who received placebo. CONCLUSIONS: A single injection of nirsevimab administered before the RSV season protected healthy late-preterm and term infants from medically attended RSV-associated lower respiratory tract infection. (Funded by MedImmune/AstraZeneca and Sanofi; MELODY ClinicalTrials.gov number, NCT03979313.).

Pango lineage designation and assignment using SARS-CoV-2 spike gene nucleotide sequences.

BACKGROUND: More than 2 million SARS-CoV-2 genome sequences have been generated and shared since the start of the COVID-19 pandemic and constitute a vital information source that informs outbreak control, disease surveillance, and public health policy. The Pango dynamic nomenclature is a popular system for classifying and naming genetically-distinct lineages of SARS-CoV-2, including variants of concern, and is based on the analysis of complete or near-complete virus genomes. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. It is therefore important to understand how much information about Pango lineage status is contained in spike-only nucleotide sequences. Here we explore how Pango lineages might be reliably designated and assigned to spike-only nucleotide sequences. We survey the genetic diversity of such sequences, and investigate the information they contain about Pango lineage status. RESULTS: Although many lineages, including the main variants of concern, can be identified clearly using spike-only sequences, some spike-only sequences are shared among tens or hundreds of Pango lineages. To facilitate the classification of SARS-CoV-2 lineages using subgenomic sequences we introduce the notion of designating such sequences to a "lineage set", which represents the range of Pango lineages that are consistent with the observed mutations in a given spike sequence. CONCLUSIONS: We find that many lineages, including the main variants-of-concern, can be reliably identified by spike alone and we define lineage-sets to represent the lineage precision that can be achieved using spike-only nucleotide sequences. These data provide a foundation for the development of software tools that can assign newly-generated spike nucleotide sequences to Pango lineage sets.

Global Molecular Epidemiology of Respiratory Syncytial Virus from the 2017-2018 INFORM-RSV Study.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017-2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of <1 year of age (81.2%), males (56.3%), and patients hospitalized for >24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017-2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.

Global molecular diversity of RSV - the "INFORM RSV" study.

BACKGROUND: Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. METHODS: The INFORM study is performed in 17 countries spanning all inhabited continents and will provide insight into the molecular epidemiology of circulating RSV strains worldwide. Sequencing of > 4000 RSV-positive respiratory samples is planned to detect temporal and geographical molecular patterns on a molecular level over five consecutive years. Additionally, RSV will be cultured from a subset of samples to study the functional implications of specific mutations in the viral genome including viral fitness and susceptibility to different monoclonal antibodies. DISCUSSION: The sequencing and functional results will be used to investigate susceptibility and resistance to novel RSV preventive or therapeutic interventions. Finally, a repository of globally collected RSV strains and a database of RSV sequences will be created.

Antiviral activity of HIV-1 integrase strand-transfer inhibitors against mutants with integrase resistance-associated mutations and their frequency in treatment-naïve individuals.

The development of resistance to human immunodeficiency virus 1 (HIV-1) integrase strand-transfer inhibitors (INSTI) has been documented; however, knowledge of the impact of pre-existing integrase (IN) mutations on INSTI resistance (INSTI-R) is still evolving. The frequency of HIV-1 IN mutations in 2177 treatment-naïve subjects was investigated, along with the INSTI susceptibility of site-directed mutant viruses containing major and minor INSTI-R mutations. Total 6 of 39 minor INSTI-R mutations (M50I, S119P/G/T/R, and E157Q) were found in >1% of IN-treatment-naïve subjects with no impact on INSTI susceptibility. When each combined with major INSTI-R mutation, M50I, S119P, and E157Q led to decreased susceptibility to elvitegravir but remained sensitive to dolutegravir and bictegravir.

Commonly Transmitted HIV-1 Drug Resistance Mutations in Reverse-Transcriptase and Protease in Antiretroviral Treatment-Naive Patients and Response to Regimens Containing Tenofovir Disoproxil Fumarate or Tenofovir Alafenamide.

Background: The presence of transmitted drug resistance mutations (TDRMs) in antiretroviral treatment (ART)-naive patients can adversely affect the outcome of ART. Methods: Resistance testing was conducted in 6704 ART-naive subjects predominantly from the United States and Europe in 9 clinical studies conducted by Gilead Sciences from 2000 to 2013. Results: The presence of TDRMs increased during this period (from 5.2% to 11.4%), primarily driven by an increase in nonnucleoside reverse-transcriptase (RT) inhibitor (NNRTI) resistance mutations (from 0.3% to 7.1%), particularly K103N/S (increase from 0.3% to 5.3%). Nucleoside/nucleotide RT inhibitor mutations were found in 3.1% of patients. Only 1 patient had K65R (0.01%) and 7 had M184V/I (0.1%), despite high use of tenofovir disoproxil fumarate (TDF), emtricitabine, and lamivudine and potential transmission of resistance to these drugs. At least 1 thymidine-analogue mutations was present in 2.7% of patients with 0.07% harboring T215Y/F and 2.7% harboring T215 revertant mutations (T215rev). Patients with the combination of M41L + L210W + T215rev showed full human immunodeficiency virus RNA suppression while receiving a TDF- or tenofovir alafenamide-containing regimen. Conclusions: There was an overall increase of TDRMs among patients enrolling in clinical trials from 2000 through 2013, driven primarily by an increase in NNRTI resistance. However, the presence of common TDRMs, including thymidine-analogue mutations/T215rev, showed no impact on response to TDF- or tenofovir alafenamide-containing regimens.

A cell-based strategy to assess intrinsic inhibition efficiencies of HIV-1 reverse transcriptase inhibitors.

During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.

Mutations in HIV-1 reverse transcriptase affect the errors made in a single cycle of viral replication.

UNLABELLED: The genetic variation in HIV-1 in patients is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Some of the mutations in reverse transcriptase (RT) that alter the deoxynucleoside triphosphate (dNTP)-binding pocket, including those that confer resistance to nucleoside/nucleotide analogs, affect dNTP selection during replication. The effects of mutations in RT on the spectrum (nature, position, and frequency) of errors made in vivo are poorly understood. We previously determined the mutation rate and the frequency of different types of mutations and identified hot spots for mutations in a lacZα (the α complementing region of lacZ) reporter gene carried by an HIV-1 vector that replicates using wild-type RT. We show here that four mutations (Y115F, M184V, M184I, and Q151M) in the dNTP-binding pocket of RT that had relatively small effects on the overall HIV-1 mutation rate (less than 3-fold compared to the wild type) significantly increased mutations at some specific positions in the lacZα reporter gene. We also show that changes in a sequence that flanks the reporter gene can affect the mutations that arise in the reporter. These data show that changes either in HIV-1 RT or in the sequence of the nucleic acid template can affect the spectrum of mutations made during viral replication. This could, by implication, affect the generation of drug-resistant mutants and immunological-escape mutants in patients. IMPORTANCE: RT is the viral enzyme that converts the RNA genome of HIV into DNA. Errors made during replication allow the virus to escape from the host's immune system and to develop resistance to the available anti-HIV drugs. We show that four different mutations in RT which are known to be associated with resistance to anti-RT drugs modestly increased the overall frequency of errors made during viral replication. However, the increased errors were not uniformly distributed; the additional errors occurred at a small number of positions (hot spots). Moreover, some of the RT mutations preferentially affected the nature of the errors that were made (some RT mutations caused an increase in insertion and deletion errors; others caused an increase in substitution errors). We also show that sequence changes in a region adjacent to a target gene can affect the errors made within the target gene.

Week 144 resistance analysis of elvitegravir/cobicistat/emtricitabine/tenofovir DF versus atazanavir+ritonavir+emtricitabine/tenofovir DF in antiretroviral-naïve patients.

OBJECTIVE: To describe baseline and emergent HIV-1 resistance to elvitegravir/ cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF) and ritonavir-boosted atazanavir/emtricitabine/tenofovir DF (ATV+RTV+FTC/TDF) in HIV-1-infected, treatment-naïve subjects through 144 weeks. METHOD: This was a randomized, double-blind, phase 3 study. HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening. Genotypic and phenotypic analyses were performed at virologic failure confirmation and retrospectively at baseline for PR, RT, and integrase (IN) for patients with virologic failure through week 144. RESULTS: In the EVG/ COBI/FTC/TDF group through week 144, HIV-1 from 8 patients (2.3%; 8/353 treated patients) developed primary IN strand transfer inhibitor (INSTI) (n = 6) and/or nucleoside RT inhibitor (NRTI) resistance substitutions (n = 7). The emergence of resistance decreased after the first year, with 5 patients developing HIV-1 resistance through week 48, 1 from weeks 48-96, and 2 from weeks 96-144. Emergent substitutions were E92Q, N155H, or Q148R (n = 2 each) and T66I or T97A (n = 1 each) in IN and M184V/I (n = 7) and K65R (n = 1) in RT. All 8 isolates had reduced susceptibility to EVG, FTC, or TDF. Virus with EVG phenotypic resistance showed cross-resistance to raltegravir. In the ATV+RTV+FTC/TDF group, HIV-1 from 2 patients (0.6%; 2/355 treated patients; both at week 144) developed the resistance substitution M184V/I in RT. CONCLUSIONS: Resistance development to EVG/COBI/FTC/TDF was infrequent (2.3%) through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

Cobicistat-boosted protease inhibitors in HIV-infected patients with mild to moderate renal impairment.

BACKGROUND: Cobicistat (COBI) is a pharmacoenhancer that optimizes systemic exposures of protease inhibitors (PIs) such as atazanavir (ATV) and darunavir (DRV). OBJECTIVE: To evaluate the efficacy and safety of switching ritonavir (RTV) to COBI in patients with creatinine clearance (CrCl) 50 to 89 mL/min who are virologically suppressed on a stable regimen containing ritonavir (RTV)-boosted ATV or DRV. Other components of the regimen remained unchanged. METHODS: A phase 3, non-comparative, open-label clinical trial. RESULTS: Seventy-three patients were enrolled. At week 48, 82% maintained virologic suppression. No emergent resistance developed. Serious adverse events (AEs) occurred in 7%, and study drug discontinuation due to AEs occurred in 10% (7 patients). There were 2 renal discontinuations and no cases of proximal renal tubulopathy. Small reductions in CrCl (median [IQR]) were observed as early as week 2, after which they were nonprogressive through week 48 (-3.8 [-9 to 0.8]). Changes in CrCl by baseline CrCl (< 70 vs ≥ 70) were -1.1 [-6.5 to 6.3] versus -6.6 [-12.4 to -0.7], respectively. CONCLUSIONS: In HIV-1-infected patients with CrCl 50 to 89 mL/min switching from RTV to COBI, COBI-boosted PIs in combination with 2 nucleos(t)ide reverse transcriptase inhibitors were well-tolerated and effective in maintaining virologic suppression. The renal safety profile of COBI in this study was consistent with the long-term data in patients without renal impairment from the phase 3 studies of COBI-containing regimens.

Impact of primary elvitegravir resistance-associated mutations in HIV-1 integrase on drug susceptibility and viral replication fitness.

Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

Durability of neutralizing RSV antibodies following nirsevimab administration and elicitation of the natural immune response to RSV infection in infants.

Nirsevimab is an extended half-life monoclonal antibody specific for the prefusion conformation of the respiratory syncytial virus (RSV) F protein, which has been studied in preterm and full-term infants in the phase 2b and phase 3 MELODY trials. We analyzed serum samples collected from 2,143 infants during these studies to characterize baseline levels of RSV-specific immunoglobulin G antibodies and neutralizing antibodies (NAbs), duration of RSV NAb levels following nirsevimab administration, the risk of RSV exposure during the first year of life and the infant's adaptive immune response to RSV following nirsevimab administration. Baseline RSV antibody levels varied widely; consistent with reports that maternal antibodies are transferred late in the third trimester, preterm infants had lower baseline RSV antibody levels than full-term infants. Nirsevimab recipients had RSV NAb levels >140-fold higher than baseline at day 31 and remained >50-fold higher at day 151 and >7-fold higher at day 361. Similar seroresponse rates to the postfusion form of RSV F protein in nirsevimab recipients (68-69%) compared with placebo recipients (63-70%; not statistically significant) suggest that while nirsevimab protects from RSV disease, it still allows an active immune response. In summary, nirsevimab provided sustained, high levels of NAb throughout an infant's first RSV season and prevented RSV disease while allowing the development of an immune response to RSV.

Molecular and phenotypic characteristics of RSV infections in infants during two nirsevimab randomized clinical trials.

Nirsevimab is a monoclonal antibody that binds to the respiratory syncytial virus (RSV) fusion protein. During the Phase 2b (NCT02878330) and MELODY (NCT03979313) clinical trials, infants received one dose of nirsevimab or placebo before their first RSV season. In this pre-specified analysis, isolates from RSV infections were subtyped, sequenced and analyzed for nirsevimab binding site substitutions; subsequently, recombinant RSVs were engineered for microneutralization susceptibility testing. Here we show that the frequency of infections caused by subtypes A and B is similar across and within the two trials. In addition, RSV A had one and RSV B had 10 fusion protein substitutions occurring at >5% frequency. Notably, RSV B binding site substitutions were rare, except for the highly prevalent I206M:Q209R, which increases nirsevimab susceptibility; RSV B isolates from two participants had binding site substitutions that reduce nirsevimab susceptibility. Overall, >99% of isolates from the Phase 2b and MELODY trials retained susceptibility to nirsevimab.

Nirsevimab binding-site conservation in respiratory syncytial virus fusion glycoprotein worldwide between 1956 and 2021: an analysis of observational study sequencing data.

BACKGROUND: Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021. METHODS: We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank. FINDINGS: We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40·0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1·0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins. INTERPRETATION: The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time. FUNDING: AstraZeneca and Sanofi.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans.

Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication.

There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZalpha reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 x 10(-5) mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZalpha, the sites favored for mutations (hot spots) in lacZalpha depended on which strand of lacZalpha was present in the viral RNA. The pattern of hot spots seen in lacZalpha in vivo did not match any of the published data obtained when purified RT was used to copy lacZalpha in vitro.

The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site.

BACKGROUND: The p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51 downward arrowRNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness was restored after repeated passage of mutant viruses, due to reversion of the mutated sequences to wild-type. However, in one case, the recovered virus retained the mutated p51 downward arrowRNH cleavage site but also developed an additional mutation, T477A, distal to the cleavage site. In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT. RESULTS: While the T477A mutation arose during serial passage only with the F440V mutant background, introduction of this substitution into a variety of RT p51 downward arrowRNH cleavage site lethal mutant backgrounds was able to restore substantial infectivity and normal RT processing to these mutants. T477A had no phenotypic effect on wild-type HIV-1. We also evaluated the impact of T477A on the kinetics of intravirion Gag-Pol polyprotein processing of p51 downward arrowRNH cleavage site mutants using the protease inhibitor ritonavir. Early processing intermediates accumulated in p51 downward arrowRNH cleavage site mutant viruses, whereas introduction of T477A promoted the completion of processing and formation of the fully processed RT p66/p51 heterodimer. CONCLUSIONS: This work highlights the extraordinary plasticity of HIV-1 in adapting to seemingly lethal mutations that prevent RT heterodimer formation during virion polyprotein maturation. The ability of T477A to restore RT heterodimer formation and thus intravirion stability of the enzyme may arise from increased conformation flexibility in the RT p51 downward arrowRNH cleavage site region, due to loss of a hydrogen bond associated with the normal threonine residue, thereby enabling proteolytic cleavage near the normal RT p51 downward arrowRNH cleavage site.