New Lidocaine-Based Pharmaceutical Cocrystals: Preparation, Characterization, and Influence of the Racemic vs. Enantiopure Coformer on the Physico-Chemical Properties

This study describes the preparation, characterization, and influence of the enantiopure vs. racemic coformer on the physico-chemical properties of a pharmaceutical cocrystal. For that purpose, two new 1:1 cocrystals, namely lidocaine:dl-menthol and lidocaine:d-menthol, were prepared. The menthol racemate-based cocrystal was evaluated by means of X-ray diffraction, infrared spectroscopy, Raman, thermal analysis, and solubility experiments. The results were exhaustively compared with the first menthol-based pharmaceutical cocrystal, i.e., lidocaine:l-menthol, discovered in our group 12 years ago. Furthermore, the stable lidocaine/dl-menthol phase diagram has been screened, thoroughly evaluated, and compared to the enantiopure phase diagram. Thus, it has been proven that the racemic vs. enantiopure coformer leads to increased solubility and improved dissolution of lidocaine due to the low stable form induced by menthol molecular disorder in the lidocaine:dl-menthol cocrystal. To date, the 1:1 lidocaine:dl-menthol cocrystal is the third menthol-based pharmaceutical cocrystal, after the 1:1 lidocaine:l-menthol and the 1:2 lopinavir:l-menthol cocrystals reported in 2010 and 2022, respectively. Overall, this study shows promising potential for designing new materials with both improved characteristics and functional properties in the fields of pharmaceutical sciences and crystal engineering

An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus

Despite the importance of phages in driving horizontal gene transfer (HGT) among pathogenic bacteria, the underlying molecular mechanisms mediating phage adsorption to S. aureus are still unclear. Phage φ11 is a siphovirus with a high transducing efficiency. Here, we show that the tail protein Gp45 localized within the φ11 baseplate. Phage φ11 was efficiently neutralized by anti-Gp45 serum, and its adsorption to host cells was inhibited by recombinant Gp45 in a dose-dependent manner. Flow cytometry analysis demonstrated that biotin-labelled Gp45 efficiently stained the wild-type S. aureus cell but not the double knockout mutant ΔtarM/S, which lacks both α- and β-O-GlcNAc residues on its wall teichoic acids (WTAs). Additionally, adsorption assays indicate that GlcNAc residues on WTAs and O-acetyl groups at the 6-position of muramic acid residues in peptidoglycan are essential components of the φ11 receptor. The elucidation of Gp45-involved molecular interactions not only broadens our understanding of siphovirus-mediated HGT, but also lays the groundwork for the development of sensitive affinity-based diagnostics and therapeutics for S. aureus infection

In Vivo Binding and Retention of CD4-Specific DARPin 57.2 in Macaques

The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 2021(1) and has multiple mutations in its spike protein(2). Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways(3) demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems

Broadly neutralizing antibodies against sarbecoviruses generated by immunization of macaques with an AS03-adjuvanted COVID-19 vaccine

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus-pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron’s evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis

Spread of a SARS-CoV-2 variant through Europe in the summer of 2020.

Following its emergence in late 2019, the spread of SARS-CoV-21,2 has been tracked by phylogenetic analysis of viral genome sequences in unprecedented detail3–5. Although the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, travel within Europe resumed in the summer of 2020. Here we report on a SARS-CoV-2 variant, 20E (EU1), that was identified in Spain in early summer 2020 and subsequently spread across Europe. We find no evidence that this variant has increased transmissibility, but instead demonstrate how rising incidence in Spain, resumption of travel, and lack of effective screening and containment may explain the variant’s success. Despite travel restrictions, we estimate that 20E (EU1) was introduced hundreds of times to European countries by summertime travellers, which is likely to have undermined local efforts to minimize infection with SARS-CoV-2. Our results illustrate how a variant can rapidly become dominant even in the absence of a substantial transmission advantage in favourable epidemiological settings. Genomic surveillance is critical for understanding how travel can affect transmission of SARS-CoV-2, and thus for informing future containment strategies as travel resumes. © 2021, The Author(s), under exclusive licence to Springer Nature Limited

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2

Walls et al. describe a potential nanoparticle vaccine for COVID-19, made of a self-assembling protein nanoparticle displaying the SARS-CoV-2 receptor-binding domain in a highly immunogenic array reminiscent of the natural virus. Their nanoparticle vaccine candidate elicits a diverse, potent, and protective antibody response, including neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike ectodomain trimer