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Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔ G ) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications., (© 2023. The Author(s).)

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic highlights the importance of determining the breadth and durability of humoral immunity to SARS-CoV-2 mRNA vaccination. Herein, we characterize the humoral response in 27 naive and 40 recovered vaccinees. SARS-CoV-2-specific antibody and memory B cell (MBC) responses are durable up to 6 months, although antibody half-lives are shorter for naive recipients. The magnitude of the humoral responses to vaccination strongly correlates with responses to initial SARS-CoV-2 infection. Neutralization titers are lower against SARS-CoV-2 variants in both recovered and naive vaccinees, with titers more reduced in naive recipients. While the receptor-binding domain (RBD) is the main neutralizing target of circulating antibodies, Moderna-vaccinated naives show a lesser reliance on RBDs, with >25% neutralization remaining after depletion of RBD-binding antibodies. Overall, we observe that vaccination induces higher peak titers and improves durability in recovered compared with naive vaccinees. These findings have broad implications for current vaccine strategies deployed against the SARS-CoV-2 pandemic., Competing Interests: E.J.A. has consulted for Pfizer, Sanofi Pasteur, Janssen, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc., and Sanofi Pasteur. His institution has also received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. M.S.S. serves on the advisory board for Moderna., (© 2022 The Author(s).)

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

One hundred cases of tetanus were treated in the intensive care unit of Leeds General Infirmary during during 1961--1977. In 90 patients disease was severe enough to require paralysis and artificial ventilation; a further three needed tracheostomy but not paralysis; and in seven the condition was mild, requiring sedation only. Ten patients died, but all deaths were attributable to complications of treatment rather than to the disease itself and were theoretically avoidable. No evidence of permanent neurological damage from tetanus was found. In 65 patients a wound was the source of infection, most of which were minor, often receiving no medical attention. In five patients the source was probably varicose ulcers of the leg. Such patients, if lacking immunity, are at risk, particularly when farming or gardening. Only two patients had received a full course of tetanus toxoid, in one case eight years and in the other three years before injury; neither received a booster dose at the time of injury and in the second tetanus was mild. Of the 35 patients who did seek medical attention at the time of wounding, 21 received prophylactic antibiotics and 25 tetanus toxoid, but none received equine or human antitetanus serum. Early resort to tracheostomy and paralysis in severe tetanus resulted in a favourable mortality of 10% in the series. To implement such a policy full intensive-care facilities, with a trained nurse to care for each patient, must be available at all times.

A four-way, double-blind, prospective trial of treatment of abscesses by incision, curettage, and primary closure with and without antibiotic cover (clindamycin injection before operation or capsules after operation, or both) was conducted. There was no appreciable difference in mean healing time between the patients given both the antibiotic injection and the antibiotic capsules and those given the injection and placebo capsules, whereas healing times in those given the placebo injection and antibiotic capsules or placebo only were appreciably longer. Four of the patients who were not given the antibiotic injection developed bacteraemia; one patient who was given the antibiotic injection also developed a bacteraemia, but this was caused by clindamycin-resistant bacteria. These results show that a single injection of an effective antibiotic before operation is sufficient to protect the patient against bacteraemia and permit optimum healing.

A white woman of thirty-two years presented with tuberculous osteitis of the third metacarpal head, secondary to previously undetected active pulmonary disease. The problems of diagnosis are illustrated by review of her history, and the literature is discussed.

A simple agglutination test for plasma immunoreactive lipase has been evaluated in 101 patients. A positive agglutination reaction was observed if, and only if, plasma lipase exceeded 300 micrograms/l. The test was positive in all 23 patients with acute pancreatitis who were tested on the day of admission to hospital, in one patient with a pancreatitic pseudocyst and in one patient with hyperamylasaemia and hyperlipasaemia due to a perforated duodenal ulcer. The simplicity and reliability of this test make it suitable as an urgent diagnostic investigation for acute pancreatitis.

A follow-up study of 50 patients treated in the tetanus unit of the General Infirmary at Leeds from 1961 to 1977 showed that 29 patients had regained normal health, nine were still improving, but 12 considered that their health had been permanently impaired. In only two of these 12, however, was the deterioration serious and unequivocally linked to their illness and treatment. All but five of those previously working had returned to full employment. Sixteen patients had unpleasant memories of the illness and treatment, 19 had psychological after effects, and one suffered serious damage to mental function. Little evidence was found of pulmonary dysfunction after the disease and its treatment, and although all the patients showed a tracheal stricture of some degree, few had directly associated disabilities. The cause of stricture is multifactorial, the surgical technique being only one. A formal surgical closure of the trachea (if a flap has been used) and of the tissue of the neck in layers should lessen residual disability from the scar and strictured areas.

A urinary test strip for amylase (Rapignost-Amylase) was compared with plasma amylase assay in samples of urine and plasma collected on the day of admission to hospital from 23 patients with acute pancreatitis and 38 patients with other causes of acute abdominal pain. Plasma amylase was greater than 1200 IU/l in 24 patients (23 with pancreatitis and 1 with a perforated duodenal ulcer) and all were Rapignost-Amylase positive. Twenty-nine of the remaining patients were Rapignost-Amylase negative, but there were eight "false positives' with plasma amylase levels of 86-474 IU/l. The Rapignost-Amylase test is of potential value to screen for clinically occult acute pancreatitis.

Pus from 84 patients with subcutaneous abscesses was examined for aerobic and anaerobic bacteria: organisms were recovered from 70 (83.3 per cent). In 13 no organisms were seen in the Gram-stained smears and the cultures showed no bacterial growth. Staphylococcus aureus was the most prevalent organism (n = 44), isolated in 43 in pure culture, in marked contrast to the anaerobic organisms which almost invariably were associated with mixed cultures. All patients were treated by the primary suture method. Half of them were not given preoperative antibiotics and 3 developed bacteraemia and 1 septicaemia. Blood isolates were of S. aureus of the same phage type as pus isolates. The other patients received one infection of 300 mg of clindamycin phosphate 30 min to 1 h before surgery. From this group only one patient, with a perianal abscess, developed Escherichia coli bacteraemia. The levels of clindamycin in the abscess aspirate were inhibitory for S. aureus, bacteroides, streptococci and other Gram-positive bacteria but not for E. coli.

We assessed the value of measuring serotonin (5-hydroxytryptamine) in plasma (by HPLC) in the diagnosis of acute appendicitis. Values for patients with subsequently confirmed appendicitis (11-145 nmol/L, median 70 nmol/L) significantly (P congruent to 0.005) exceeded those for patients with abdominal pain in whom appendicitis was only a possible diagnosis (2-45 nmol/L, median 20 nmol/L). The results for appendicitis patients were bimodally distributed, with low results found in patients where surgery revealed gangrenous appendicitis with little viable appendicular tissue. We conclude that measuring serotonin may be of value in confirming or excluding the diagnosis of early acute appendicitis where the physical signs are equivocal, and thus helps reduce unnecessary appendectomies. However, serotonin is of little help in diagnosing gangrenous appendicitis, where physical signs are more likely to be clearcut.