The potential emergence of SARS-CoV-2 Spike (S) escape mutants is a threat to reduce the efficacy of existing vaccines and neutralizing antibody (nAb) therapies. An understanding of the antibody/S escape mutation landscape is urgently needed to preemptively address this threat. Here we describe a rapid method to identify escape mutants for nAbs targeting the S receptor binding site. We identified escape mutants for five nAbs, including three from the public germline class VH3-53 elicited by natural COVID-19 infection. Escape mutations predominantly mapped to the periphery of the ACE2 recognition site on the RBD with K417, D420, Y421, F486, and Q493 as notable hotspots. We provide libraries, methods, and software as an openly available community resource to accelerate new therapeutic strategies against SARS-CoV-2.

Q fever (Coxiella burnetii) is a zoonotic disease of increasing public health importance. The objective of this study was to estimate the prevalence of, and risk factors associated with, exposure to C. burnetii in cattle in the Republic of Ireland. Bulk-tank milk samples from 290 dairy herds and 1659 sera from 332 dairy and beef herds, randomly sampled, were tested by indirect ELISA to detect antibodies to C. burnetii. In total, 37·9% of bulk-milk sample herds and 1·8% of sera (from 6·9% of herds) were antibody positive. Of risk factors tested using logistic regression analysis, only large herd size (bulk-milk analysis) and dairy breed (serum analysis) significantly increased the odds of being positive for antibodies to C. burnetii. Herds with positive milk or serum samples were randomly distributed throughout the Republic of Ireland and no clustering was observed. The use of an ELISA to test bulk-milk samples collected by randomized stratified sampling is a cost-effective method by which national herd prevalence can be estimated by active surveillance.

User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.

Generating combinatorial libraries of specific sets of mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well as functional antibody and enzyme engineering. Here we present optimization of a combinatorial mutagenesis method involving template-based nicking mutagenesis, which allows for the generation of libraries with >99% coverage for tens of thousands of user-defined variants. The non-optimized method resulted in low library coverage, which could be rationalized by a model of oligonucleotide annealing bias resulting from the nucleotide mismatch free-energy difference between mutagenic oligo and template. The optimized method mitigated this thermodynamic bias using longer primer sets and faster annealing conditions. Our updated method, applied to two antibody fragments, delivered between 99.0% (32451/32768 library members) to >99.9% coverage (32757/32768) for our desired libraries in 2 days and at an approximate 140-fold sequencing depth of coverage.

An immunoperoxidase technique for the localisation of leptospires in sections of formalin fixed paraffin embedded kidney tissue is described. The procedure utilises a two-layered antibody sandwich with rabbit anti-leptospiral immunoglobin. Using antiserum to specific leptospiral serovars the presence and distribution of specific serovar in the tissue could be determined. The technique was also used to detect leptospires of given serovars in smears made from infected tissues and fluids. There was good correlation between culture results and results of the immunoperoxidase staining method on kidneys infected with leptospires. The diagnostic possibilities of the technique on formalin fixed tissue specimens are discussed.