AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Early loosening and secondary dislocation due to a broken trochanteric osteotomy wire following a Charnley total hip arthroplasty: a case report.

We report a case of interposition of a broken trochanteric wire in the hip joint. This caused early wear of the prosthesis and dislocation of the Charnley total hip arthroplasty. The patient was treated with a revision total hip arthroplasty. This rare complication should be taken into consideration when performing a trochanteric osteotomy fixation with wiring in Charnley total hip arthroplasty.

Mycobacterium tuberculosis ClpC1: characterization and role of the N-terminal domain in its function.

Caseinolytic protein, ClpC is a general stress protein which belongs to the heat shock protein HSP100 family of molecular chaperones. Some of the Clp group proteins have been identified as having a role in the pathogenesis of many bacteria. The Mycobacterium tuberculosis genome demonstrates the presence of a ClpC homolog, ClpC1. M. tuberculosis ClpC1 is an 848-amino acid protein, has two repeat sequences at its N-terminus and contains all the determinants to be classified as a member of the HSP100 family. In this study, we overexpressed, purified and functionally characterized M. tuberculosis ClpC1. Recombinant M. tuberculosis ClpC1 showed an inherent ATPase activity, and prevented protein aggregation. Furthermore, to investigate the contribution made by the N-terminal repeats of ClpC1 to its functional activity, two deletion variants, ClpC1Delta1 and ClpC1Delta2, lacking N-terminal repeat I and N-terminal repeat I along with the linker between N-terminal repeats I and II, respectively were generated. Neither deletion affected the ATPase activity. However, ClpC1Delta1 was structurally altered, less stable and was unable to prevent protein aggregation. Compared with wild-type protein, ClpC1Delta2 was more active in preventing protein aggregation and displayed higher ATPase activity at high pH values and temperatures. The study demonstrates that M. tuberculosis ClpC1 manifests chaperone activity in the absence of any adaptor protein and only one of the two N-terminal repeats is sufficient for the chaperone activity. Also, an exposed repeat II makes the protein more stable and functionally more active.

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli.

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.

First metatarsophalangeal joint arthrodesis: a new technique of internal fixation by using memory compression staples.

A prospective clinical study of first metatarsophalangeal joint arthrodesis using memory compression staples is presented. In 27 patients, 30 feet underwent surgery. There were 24 women and 3 men, with a mean age of 61.2 years. Two memory compression staples were used at right angles to each other to achieve compression at the fusion site. Postoperatively, patients were allowed full weightbearing in a rigid-soled shoe. Subjective assessment was performed with a standard questionnaire, which included questions regarding level of pain, ambulation, and patient satisfaction. Objective assessment was performed by a clinical and included a radiographic examination. There was a postoperative reduction in the pain score from 4.6 to 1.6 (P < .0001). Ambulation ability improved from 4 to 2.5 (P < .0001). Patients reported 86.6% excellent to good results, and 96.7% achieved radiographic fusion at an average 8.2 weeks. The only significant postoperative complication was a single nonunion. The authors advocate memory compression staples for the internal fixation of first metatarsophalangeal joint arthrodesis. The implant is low profile, and postoperative cast immobilization is not required. The use of this device has a predictable success rate comparable to previously reported methods.

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis.

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.

PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response.

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein.

Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India.

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.