The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

The biological and clinical significance of emerging SARS-CoV-2 variants.

The past several months have witnessed the emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic. These variants can increase rates of virus transmission and/or increase the risk of reinfection and reduce the protection afforded by neutralizing monoclonal antibodies and vaccination. These variants can therefore enable SARS-CoV-2 to continue its spread in the face of rising population immunity while maintaining or increasing its replication fitness. The identification of four rapidly expanding virus lineages since December 2020, designated variants of concern, has ushered in a new stage of the pandemic. The four variants of concern, the Alpha variant (originally identified in the UK), the Beta variant (originally identified in South Africa), the Gamma variant (originally identified in Brazil) and the Delta variant (originally identified in India), share several mutations with one another as well as with an increasing number of other recently identified SARS-CoV-2 variants. Collectively, these SARS-CoV-2 variants complicate the COVID-19 research agenda and necessitate additional avenues of laboratory, epidemiological and clinical research.

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2.

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating.

SARS-CoV-2 evolution during treatment of chronic infection.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies.

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.

HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation.

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.

Population Effectiveness of Dolutegravir Implementation in Uganda - A Prospective Observational Cohort Study (DISCO): 48-week Results.

BACKGROUND: Tenofovir/lamivudine/dolutegravir (TLD) is the preferred first-line antiretroviral therapy (ART) regimen for people with HIV (PWH), including those who were previously virologically suppressed on non-nucleoside reverse transcriptase inhibitors (NNRTIs). We sought to estimate the real-world effectiveness of the TLD transition in Ugandan public-sector clinics. METHODS: We conducted a prospective cohort study of PWH ≥18 years who were transitioned from NNRTI-based ART to TLD. Study visits were conducted on the day of TLD transition and 24- and 48- weeks later. The primary endpoint was viral suppression (<200 copies/mL) at 48-weeks. We collected blood for retrospective viral load (VL) assessment and conducted genotypic resistance tests for specimens with VL >500 copies/mL. RESULTS: We enrolled 500 participants (median age of 47 years; 41% women). At 48-weeks after TLD transition, 94% of participants were in care with a VL <200 copies/mL (n = 469/500); 2% (n = 11/500) were lost from care or died; and only 2% (n = 9/500) had a VL >500 copies/mL. No incident resistance to DTG was identified. Few participants (2%, n = 9/500) discontinued TLD due to adverse events. CONCLUSIONS: High rates of viral suppression, high tolerability, and lack of emergent drug resistance support use of TLD as the preferred first-line regimen in the region.

A compound eye-like morphology formed through hexagonal array of hemispherical microparticles where an alkyl-fullerene derivative self-assembled at atmosphere-sealed air/water interface.

Self-assembly processes are widely used in nature to form hierarchically organized structures, prompting us to investigate such processes at the macroscopic scale. We report an unprecedented approach toward the self-assembly of alkyl-fullerene (C60) derivatives into a hexagonal array of hemispherical microparticles akin to the morphology of a compound eye. The method includes casting solvated alkyl-C60compound on an air/water interface followed by controlled evaporation of the solvent under atmosphere-sealed conditions. This leads to the formation of a thin film floating on water with a diameter of up to 1.3 centimeters and exhibiting a hexagonally-packed hemispherical structure with a diameter of approximately 38µm. Various measurements of the formed film reveal that amorphousness is necessary for suppressing uncontrollable crystallization, which affects the microparticle size and film formation mechanism. We tested the feasibility of this approach for the self-assembly of a relatively common C60derivative, [6,6]-phenyl-C61-butyric acid methyl ester (PC61BM), resulting in the formation of a film with a similar pattern of hexagonally-packed larger microparticles approximately 152µm in size of diameter.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Limited emergence of resistance to integrase strand transfer inhibitors (INSTIs) in ART-experienced participants failing dolutegravir-based antiretroviral therapy: a cross-sectional analysis of a Northeast Nigerian cohort.

BACKGROUND: Due to the high prevalence of resistance to NNRTI-based ART since 2018, consolidated recommendations from the WHO have indicated dolutegravir as the preferred drug of choice for HIV treatment globally. There is a paucity of resistance outcome data from HIV-1 non-B subtypes circulating across West Africa. AIMS: We characterized the mutational profiles of persons living with HIV from a cross-sectional cohort in North-East Nigeria failing a dolutegravir-based ART regimen. METHODS: WGS of plasma samples collected from 61 HIV-1-infected participants following virological failure of dolutegravir-based ART were sequenced using the Illumina platform. Sequencing was successfully completed for samples from 55 participants. Following quality control, 33 full genomes were analysed from participants with a median age of 40 years and median time on ART of 9 years. HIV-1 subtyping was performed using SNAPPy. RESULTS: Most participants had mutational profiles reflective of exposure to previous first- and second-line ART regimens comprised NRTIs and NNRTIs. More than half of participants had one or more drug resistance-associated mutations (DRMs) affecting susceptibility to NRTIs (17/33; 52%) and NNRTIs (24/33; 73%). Almost a quarter of participants (8/33; 24.4%) had one or more DRMs affecting tenofovir susceptibility. Only one participant, infected with HIV-1 subtype G, had evidence of DRMs affecting dolutegravir susceptibility-this was characterized by the T66A, G118R, E138K and R263K mutations. CONCLUSIONS: This study found a low prevalence of resistance to dolutegravir; the data are therefore supportive of the continual rollout of dolutegravir as the primary first-line regimen for ART-naive participants and the preferred switch to second-line ART across the region. However, population-level, longer-term data collection on dolutegravir outcomes are required to further guide implementation and policy action across the region.

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy.

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.

Highly Soluble Ambipolar anti-Perylene-3,4 : 9,10-bis(benzimidazole)s Stabilize a Room-Temperature Columnar Hexagonal Phase.

A new series of highly soluble perylene anti-bis(4,5-dialkoxybenzimidazole)s bearing branched flexible chains stabilizing room temperature columnar hexagonal phase and with balanced ambipolar charge carrier mobility is reported for the first time. Only the anti isomer was successfully separated and characterized. These compounds have a high extinction coefficient, small optical band gap and wide absorption range, thus making them a promising class of ambipolar organic semiconductors capable of self-organizing.

DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages.

We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs.

Deep sequencing of HIV-1 reveals extensive subtype variation and drug resistance after failure of first-line antiretroviral regimens in Nigeria.

BACKGROUND: Deep sequencing could improve understanding of HIV treatment failure and viral population dynamics. However, this tool is often inaccessible in low- and middle-income countries. OBJECTIVES: To determine the genetic patterns of resistance emerging in West African HIV-1 subtypes during first-line virological failure, and the implications for future antiretroviral options. PATIENTS AND METHODS: Participants were selected from a Nigerian cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy. Whole HIV-1 genome sequences were generated from first-line virological failure samples with Illumina MiSeq. Mutations detected at ≥2% frequency were analysed and compared by subtype. RESULTS: HIV-1 sequences were obtained from 101 participants (65% female, median age 30 years, median 32.9 months of nevirapine- or efavirenz-based ART). Thymidine analogue mutations (TAMs) were detected in 61%, other core NRTI mutations in 92% and NNRTI mutations in 99%. Minority variants (<20% frequency) comprised 18% of all mutations. K65R was more prevalent in CRF02_AG than G subtypes (33% versus 7%; P = 0.002), and ≥3 TAMs were more common in G than CRF02_AG (52% versus 24%; P = 0.004). Subtype G viruses also contained more RT cleavage site mutations. Cross-resistance to at least one of the newer NNRTIs, doravirine, etravirine or rilpivirine, was predicted in 81% of participants. CONCLUSIONS: Extensive drug resistance had accumulated in people with West African HIV-1 subtypes, prior to second-line ART. Deep sequencing significantly increased the detection of resistance-associated mutations. Caution should be used if considering newer-generation NNRTI agents in this setting.

Tetramethyl Succinonitrile as a Solid Plasticizer in a Poly(ethylene oxide)8 -LiI-I2 Solid Polymer Electrolyte.

Dye-sensitized solar cell (DSSC) is a promising alternative to the commercially available amorphous silicon-based solar cell because of several advantageous properties. A DSSC with a fast ion conducting solid polymer electrolyte is required for the arid atmosphere of Gulf countries. In this work, a new matrix, poly(ethylene oxide)-tetramethyl succinonitrile blend to synthesize a blend-LiI-I2 solid polymer electrolyte for the DSSC application has been proposed. The tetramethyl succinonitrile is a member of plastic crystal with a solid-solid phase transition temperature (Tpc ) of ≈71 °C and melting temperature (Tm ) of ≈170.5 °C. Its molar fraction, 0.1-0.15 is sufficient enough for synthesizing a polymer electrolyte with electrical conductivity of >10-4 S cm-1 at room temperature. This electrolyte shows Vogel-Tamman-Fulcher type behavior with a low value (≈0.083 eV) of pseudo-activation energy for easy ion transport. The results of Fourier-transform infrared spectroscopy, X-ray diffractometry, and differential scanning calorimetry studies reveal the plasticizing effect of tetramethyl succinonitrile to form an amorphous phase. This electrolyte results in a ≈661% gain in short-circuit current density and thereby a ≈552% gain in the cell efficiency (≈3.5%) with respect to the DSSC prepared with the tetramethyl succinonitrile-free electrolyte.

SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.