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The SARS-CoV-2 omicron (B.1.1.529) variant, which was first identified in November, 2021, spread rapidly in many countries, with a spike protein highly diverged from previously known variants, and raised concerns that this variant might evade neutralising antibody responses. We therefore aimed to characterise the sensitivity of the omicron variant to neutralisation.For this cross-sectional study, we cloned the sequence encoding the omicron spike protein from a diagnostic sample to establish an omicron pseudotyped virus neutralisation assay. We quantified the neutralising antibody IDNeutralising antibody responses in reference sample pools sampled shortly after infection or vaccination were substantially less potent against the omicron variant than against wild-type SARS-CoV-2 (seven-fold to 42-fold reduction in IDThese data highlight the extensive, but incomplete, evasion of neutralising antibody responses by the omicron variant, and suggest that boosting with licensed vaccines might be sufficient to raise neutralising antibody titres to protective levels.European Union Horizon 2020 research and innovation programme, European and Developing Countries Clinical Trials Partnership, SciLifeLab, and the Erling-Persson Foundation.

HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV.

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciencessinglemoleculereal-time platform was used to sequence PCR amplicons derived from cDNA templates tagged withuniversalmolecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and the use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies.Author SummaryThere is a great need to understand the genetic diversity of pathogens in an accurate and timely manner, but many errors can be introduced during the sample handling and sequencing steps which may prevent accurate analyses. In some cases, the errors introduced during these steps can be indistinguishable from real genetic variation and prevent analyses from identifying true sequence variation present in the pathogen population. There are established methods which can help to prevent these types of errors, but can involve many different steps and variables, all of which must be optimized and tested together to ensure the desired effect. Here we show results from testing different methods on a set of HIV+ blood plasma samples and arrive at a streamlined laboratory protocol and bioinformatic pipeline which prevents or corrects for different types of errors that can arise in sequence datasets. These methods should be an accessible starting point for anyone wanting accurate sequencing without extensive optimizations.

Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the Beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

The recently-emerged SARS-CoV-2 B.1.1.529 variant (Omicron) is spreading rapidly in many countries, with a spike that is highly diverged from the pandemic founder, raising fears that it may evade neutralizing antibody responses. We cloned the Omicron spike from a diagnostic sample which allowed us to rapidly establish an Omicron pseudotyped virus neutralization assay, sharing initial neutralization results only 13 days after the variant was first reported to the WHO, 8 days after receiving the sample.Here we show that Omicron is substantially resistant to neutralization by several monoclonal antibodies that form part of clinical cocktails. Further, we find neutralizing antibody responses in pooled reference sera sampled shortly after infection or vaccination are substantially less potent against Omicron, with neutralizing antibody titers reduced by up to 45 fold compared to those for the pandemic founder. Similarly, in a cohort of convalescent sera prior to vaccination, neutralization of Omicron was low to undetectable. However, in recent samples from two cohorts from Stockholm, Sweden, antibody responses capable of cross-neutralizing Omicron were prevalent. Sera from infected-then-vaccinated healthcare workers exhibited robust cross-neutralization of Omicron, with an average potency reduction of only 5-fold relative to the pandemic founder variant, and some donors showing no loss at all. A similar pattern was observed in randomly sampled recent blood donors, with an average 7-fold loss of potency. Both cohorts showed substantial between-donor heterogeneity in their ability to neutralize Omicron. Together, these data highlight the extensive but incomplete evasion of neutralizing antibody responses by the Omicron variant, and suggest that increasing the magnitude of neutralizing antibody responses by boosting with unmodified vaccines may suffice to raise titers to levels that are protective.

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Vertical profiles of black carbon (BC) and other light-absorbing impurities were measured in seasonal snow and permanent snowfields in the Chilean Andes during Austral winters 2015 and 2016, at 22 sites between latitudes 18°S and 41°S. The samples were analyzed for spectrally-resolved visible light absorption. For surface snow, the average mass mixing ratio of BC was 15 ng/g in northern Chile (18–33°S), 28 ng/g near Santiago (a major city near latitude 33°S, where urban pollution plays a significant role), and 13 ng/g in southern Chile (33–41°S). The regional average vertically-integrated loading of BC was 207 µg/m2 in the north, 780 µg/m2 near Santiago, and 2500 µg/m2 in the south, where the snow season was longer and the snow was deeper. For samples collected at locations where there had been no new snowfall for a week or more, the BC concentration in surface snow was high (~10–100 ng/g) and the sub-surface snow was comparatively clean, indicating the dominance of dry deposition of BC. Mean albedo reductions due to light-absorbing impurities were 0.0150, 0.0160, and 0.0077 for snow grain radii of 100 µm for northern Chile, the region near Santiago, and southern Chile; respective mean radiative forcings for the winter months were 2.8, 1.4, and 0.6 W/m2. In northern Chile, our measurements indicate that light-absorption by impurities in snow was dominated by dust rather than BC.

SARS-CoV-2 Variants of Concern (VOCs) with resistance to neutralizing antibodies are threatening to undermine vaccine efficacy. Vaccination and infection have led to widespread humoral immunity against the pandemic founder (Wu-Hu-1). Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet clear whether heterotypic boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. We show, in macaques immunized with Wu-Hu-1 spike, that a single dose of adjuvanted beta variant receptor binding domain (RBD) protein broadens neutralizing antibody responses to heterologous VOCs. Passive transfer of plasma sampled after Wu-Hu-1 spike immunization only partially protects K18-hACE2 mice from lethal challenge with a beta variant isolate, whereas plasma sampled following heterotypic RBD boost protects completely against disease., Graphical Abstract, The emergence and spread of antibody-resistant SARS-CoV-2 Variants of Concern (VOCs) threatens to diminish vaccine efficacy. Sheward et al. show, in rhesus macaques and K18-hACE2 mice, that reduced vaccine protection against VOCs can be restored by broadening antibody responses with a third, heterotypic RBD booster immunization.

The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.

For HIV, the time since infection can be estimated from sequence data for acutely infected samples. One popular approach relies on the star-like nature of phylogenies generated under exponential population growth, and the resulting Poisson distribution of mutations away from the founding variant. However, real-world complications, such as APOBEC hypermutation and multiple-founder transmission, present a challenge to this approach, requiring data curation to remove these signals before reasonable timing estimates may be obtained. Here we suggest a simple alternative approach that derives the timing estimate not from the entire mutational spectrum but from the proportion of sequences that have no mutations. This can be approximated quickly and is robust to phenomena such as multiple founder transmission and APOBEC hypermutation. Our approach is Bayesian, and we adopt a conjugate prior to obtain closed form posterior distributions at negligible computational expense. Using real data and simulations, we show that this approach provides accurate timing estimates and credible intervals without the inconvenience of data curation and is robust to complicating phenomena that can mislead existing approaches or cause them to fail entirely. For immediate use we provide an implementation via Google Sheets, which offers bulk analysis of multiple datasets, as well as more detailed individual-donor analyses. For inclusion in data processing pipelines we provide implementations in three languages: Julia, R, and Python.